T cell receptor knock out engineered immune cells, endowed with chimeric antigen receptors binding to cd123 for the treatment of relapsed/refractory acute myeloid lymphoma or blastic plasmacytoid dendritic cell neoplasm

ABSTRACT

The present invention relates to a TCR KO—or TCR KO and dCK KO—engineered immune cells expressing a Chimeric Antigen Receptors (CAR) specific for CD123 that is a recombinant chimeric protein able to redirect immune cell specificity and reactivity toward CD123-expressing cells, and more particularly in which extracellular ligand binding is a scFV derived from a CD123 monoclonal antibody, conferring specific immunity against CD123 positive cells. The engineered immune cells endowed with such CD123 CARs are particularly suited for treating relapse refractory AML and blastic plasmacytoid dendritic cell neoplasm and for use as a treatment before bone marrow transplantation.

FIELD OF THE INVENTION

The present invention relates generally to a TCR gene Knock out T cellengineered to express a Chimeric Antigen Receptor (CAR) specific forinterleukin 3 receptor alpha chain (IL-3Ra, cluster of differentiation123 CD123) and their use, e.g., for the treatment of a disease or acondition associated with expression of IL-3Ra, CD123, namely acutemyeloid leukemia (AML) and Blastic plasmacytoid dendritic cell neoplasm(BPDCN).

The present invention relates to a T cell receptor (TCRalpha or betagene) and/or human deoxycytidine kinase (dCK gene or dck gene) Knock out(KO) immune T cells engineered to express:

-   -   a Chimeric Antigen Receptor (CAR) specific for the cluster of        differentiation 123 (CD123) comprising at least one        extracellular ligand binding domain from Klon43, optionally        humanized, a hinge, an intracellular domain and a costimulatory        domain,    -   a suicide domain, optionally inserted into a said hinge,        said CD123 CAR being specific for human CD123 and conferring a        specific immunity against CD123 positive cells,        in one embodiment; this object for its used for the treatment of        acute myeloid leukemia AML, or of a complication of AML is        provided,        in one embodiment; this object is provided for the treatment of        Blastic plasmacytoid dendritic cell neoplasm (BPDCN).        in one embodiment; this object is provided as a treatment before        bone marrow transplant as a bridge of transplant.

The engineered immune cells endowed with the CD123 CARs according to theinvention show high efficiency in view of treating lymphomas andleukemias as compared to previous CD123 CAR, and can be used in thepresence of purine analogue with less side effects than previoustreatments in patients.

BACKGROUND OF THE INVENTION

Induction treatments for lymphoproliferative diseases such as leukemiaand in particular for acute myeloid leukemia (AML) have remained largelyunchanged for nearly 50 years. Such standard induction chemotherapy caninduce complete remissions, but many patients eventually relapse andsuccumb to the disease, calling for the development of noveltherapeutics for AML, in particular for relapsed refractory AML.

Similar observations were reported for aggressive lymphoproliferativediseases such as BPDCN. These diseases remain of poor prognosis.Immunophenotyping of these cancerous cells have revealed that theinterleukin 3 receptor alpha chain (IL-3Rα; CD123—NCBI reference:NP_001254642) is a potential immunotherapeutic target since it isover-expressed on these tumor cells compared to normal cells.Additionally, two phase I trials for CD123-specific therapeutics havebeen completed with both drugs displaying good safety profiles(ClinicalTrials.gov ID: NCT00401739 and NCT00397579). Unfortunately,these CD123 targeting drugs had limited efficacy suggesting thatalternative, and more potent and specific therapies targeting CD123 arerequired to observe anti-leukemic activity.

A possibly more potent alternative therapy for the treatment of Leukemiacould be the use of immune cells expressing chimeric antigen receptors(CARs) that selectively direct immune cell specificity towards cellsurface tumor associated antigens (TAAs) in an MHC-independent manner(Jena, Dotti et al. 2010) and destroy them.

CARs are synthetic receptors consisting of a targeting moiety that isassociated with one or more signaling domains in a single or multiplefusion molecule(s). In general, the binding moiety of a CAR consists ofan antigen-binding domain of a single-chain antibody (scFv), comprisingthe light and variable fragments of a monoclonal antibody joined by aflexible linker. Binding moieties based on receptor or ligand domainshave also been used successfully. The signaling domains for firstgeneration CARs are derived from the cytoplasmic region of the CD3zetaor the Fc receptor gamma chains. First generation CARs have been shownto successfully redirect T cell cytotoxicity, however, they failed toprovide prolonged expansion and anti-tumor activity in vivo. Signalingdomains from co-stimulatory molecules including OX-40 (CD134), and 4-1BB(CD137) have been added alone or in combination to enhance survival andincrease proliferation of CAR modified T cells in vitro. However, notall combination of domains results in a CAR that can direct immune cellsto target cells in vivo.

In mouse models of human cancer, CARs can redirected the CAR-expressingcells against antigens expressed at the surface of tumor cells with anefficiency dependent on the nature and length of each domain (CondomineM. et al., 2015 Plos One 10(6):e0130518). So far autologous transfer ofCAR-expressing specific T cells, alone, has been shown to be successfulin treating specific forms of cancer despite several side effects suchas cytokine storm, non specific destruction of cell populations, orunwanted specific immune reactions (Park, Rosenberg et al. 2011). It isnot known whether allogenic CD123 CAR engineered T cells can be usedsafely and efficiently for the treatment of AML (ClinicalTrials.gov ID:NCT02159495).

Thus, to broaden the population of patients that may benefit suchtreatments so called allogenic T cells expressing CAR have been preparedfor their use in human suffering cancer. in that case, immune cells areisolated from healthy donors, engineered, and then used as a treatmentin several different selected patients in need thereof.

For that purpose and to reduce the risk of potential graft versus hostdisease, selected genes were carefully knocked out to provide engineeredCAR-expressing immune cells with reduced expression of moleculesinvolved in the immune response, for example that of MHC molecules orsubunits of the TCR molecules. The extent to which such engineered cellsstill proliferate and survive in any hosts remains largely unknown,especially in patients already treated with chemotherapy agents.

The use of such CAR expressing immune T cell targeting CD123 incombination with cytotoxic chemotherapy agents as a treatment usuallyemployed as anti-cancer treatments remains a problem as anti-cancertreatments also affect the proliferation and/or survival of T cells.

Thus, there is also a need of developing T cells targeting CD123 thatwould be specific efficient and compatible with the use of drugs, inparticular of anti-cancer chemotherapies, such as those affecting cellproliferation, that would still be able to proliferate and survive toreach their target (cancer cells).

To use “off-the-shelf” allogeneic therapeutic cells in conjunction withchemotherapy, the inventors identified means to provide allogenicengineering T-cell, less allogenic and permissive for the immune systemof patients suffering a cancer and/or already treated withchemotherapeutic agents. The therapeutic benefits are provided by thesynergistic effects between chemotherapy and immunotherapy of theclaimed objects.

The present invention is also drawn to anti-CD123 chimeric antigenreceptors (anti-CD123 CAR), which extracellular binding domain (scFv) ismodified in such a way to allow both cell sorting and cell depletion.The structure allowing this is an epitope recognized by a monoclonalantibody (named mAb-driven sorting/depletion system or epitoperecognized by a specific monoclonal antibody or mimotope) and comprisesa selected epitope inserted into the extracellular domain within thescFv and/or the hinge. This epitope has the specificity to be recognizedby a specific antibody (preferably a monoclonal antibody (mAb),optionally humanized). Given the fact that mainly the external ligandbinding domain of the CAR is modified to include the epitope, differentCAR architectures can be envisioned: single-chain or multi-chain asdisclosed in PCTUS2013/058005.

The chimeric scFv of the invention, which is formed of the VH and VLpolypeptides and the specific epitope(s) may itself have differentstructures depending on the position of insertion of the epitope and theuse of linkers (FIG. 3). The present invention also relates to theresulting method for sorting and/or depleting the engineered immunecells endowed with the modified CARs.

Several epitope-mAb couples can be used to generate such system; inparticular those already approved for medical use, such asCD20/rituximab as a non-limiting example.

Finally, the invention encompasses therapeutic methods where number,activity and survival of the engineered immune cells endowed withanti-CD123 CARs is modulated by depleting the cells by using an antibodythat directs the external ligand binding domain of said CARs.

SUMMARY OF THE INVENTION

Interleukin 3 receptor alpha chain (CD123) has been identified as beingfrequently over-expressed on Leukemia tumor cells, especially in thecase of acute myeloid leukemia (AML), compared to normal cells of thesame lineage.

The inventors have generated an immune cell engineered to express aCD123 specific CAR comprising a scFV from KLON43 antibody, a hinge fromFcγRIIIα, and intracellular domains conferring host cells the capacityto proliferate in vivo, reach CD123 target cells and alter theirsurvival, said cell comprising additional marker or suicide domainallowing their specific destruction once target cells are contained.These CD123 specific CAR are designated CD123 specific CAR or anti-CD123CAR, or 123 CAR, or “CAR of the invention” indiscriminately.

In the present invention, a specific and selective tolerogenic TCR KOCD123 CAR expressing T cell was prepared using one antibody specific forthis IL-3 receptor subunit, namely klon43 or humanized sequences derivedfrom this klon43 antibody for the treatment of patients suffering AML,B-cell lymphoproliferative disorder (BC-LPD) or BPDCN.

Concomitantly, these cells allow the destruction precancerous cellsstopping the progression and emergence of refractory/relapsed cancer.Due to their capacity to proliferate in vivo and reach tissues or nichesthese cells acts faster than cancerous cells themselves and caneradicate even aggressive lymphoproliferative disorder.

Following non-specific activation in vitro (e.g. with anti CD3/CD28coated beads and recombinant IL2), T-cells from donors have beentransformed with polynucleotides expressing CARs of the invention usingviral transduction. The T-cells were further engineered to createless-alloreactive T-cells, by disruption of a component of the T cellreceptor TCR (αβ-T-Cell receptors) to reduce Graft versus host reaction.

In a preferred embodiment, T-cells were further engineered by deletingspecific combination(s) of genes identified in table 9, to createtolerogene T cells resistant to anti-cancer drugs, to be used incombination with said classical anti-cancer drugs, namely purineanalogs.

The resulting engineered T-cells displayed reactivity against CD123positive cells showing that the CARs of the present invention contributeto antigen dependent activation, and also proliferation, of the T-cells,making them useful for immunotherapy.

The resulting engineered T-cells displayed reactivity in-vivo againstCD123 positive cells and significantly reduce the number of cancer cellsin vivo.

In a particular embodiment, several administrations of the engineeredT-cells of the invention can be performed, making them useful forimmunotherapy as a first treatment (induction), as a consolidationtreatment, as a treatment in combination with classical anticancerchemotherapy.

The polypeptides and polynucleotide sequences encoding the CARs of thepresent invention are detailed in the present specification.

The engineered immune cells of the present invention are particularlyuseful for therapeutic applications such as B-cell lymphoma or leukemiatreatments and can be selectively eliminated from the organism. R

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Schematic representation of an engineered immune cell accordingto the invention. The engineered immune cell presented in this figure isa T-cell transduced with a retroviral polypeptide encoding CAR. ThisT-cell is further engineered to allow a better and safer engraftmentinto the patient, which is part of the frame of the present invention. Xgene is a gene expressing a component of TCR (TCRalpha or TCRbeta gene),Y a gene involved into the sensitivity of T-cells to purine analogues,dCK.

FIG. 2: Schematic representation of the two CAR of the invention (123CAR) comprising a CD123 scfv, optionally humanized, a hinge from FcRIIIor CD8alpha, a transmembrane domain from CD8alpha, and two intracellulardomains from 4-1BB and CD3zeta.

FIG. 3: Shows examples of a CAR according to the invention.

L: linker between VH and VL of (GGGGS)n with n=1 to 4, preferably n=3(SEQ ID NO.: 10)

Li: linker sequence corresponding to GGGS or SGGGGS or GSGGGGS, TM:transmembrane domain.

A CD123 CAR of the invention comprising a VH from Klon 43, optionallyhumanized, a linker L, a VL from Klon 43, optionally humanized, asuicide domain (e.g. two copies of a CD20 mimotope of sequenceCPYSNPSLCS (SEQ ID NO. 161), and a copy of SEQ ID NO 169), saidmimotopes are located preferably in the scfv, a CD8 hinge or part of it,a transmembrane domain (CD8 TM) from CD8alpha, a co-stimulatory domain(4-113B) and a stimulatory domain (ITAM CD3 zeta), was prepared.

In a CD123 CAR of the invention, two copies of a CD20 mimotope ofsequence CPYSNPSLCS, linked to each other and to the VL by a linker Li,were inserted between the anti-CD123 scFv and a hinge from CD8alpha, anoptional linker LI joins the mimotopes to the hinge (SEQ ID NO 160).

Other possibilities of epitope (eg in scfv) insertion are contemplatedand illustrated as a circle in FIG. 3.

FIG. 4: Degranulation activity of different a scFv according to theinvention for one architecture (v3: CD8-hinge/CD8-transmembrane), whenCAR+ T-cells were co-cultured for 6 hours with CD123 expressing cells(RPM18226), or with cells that do not express CD123 (K562).

FIG. 5: IFN gamma release by T-cells when co-cultured for 24 h withcells expressing different levels of CD123 (KG1a or RPM18226), or withcells that do not express CD123 (K562)

FIG. 6: Dose-response of the specific cytolytic activity of CAR-T cellsin vivo in mice treated with PNA (20 mg/kg) ip.

FIG. 7: Dose-response of the specific cytolytic activity of CAR-T cellsin vivo in mice treated with PNA (20 mg/kg) ip.

TABLE 1 Functional domains SEQ ID # amino acid sequence CD8α signalpeptide SEQ ID NO. 1 MALPVTALLLPLALLLHAARP Alternative signal peptideSEQ ID NO. 2 METDTLLLWVLLLWVPGSTG FcγRIIIα hinge SEQ ID NO. 3GLAVSTISSFFPPGYQ CD8α hinge SEQ ID NO. 4 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD IgG1 hinge SEQ ID NO. 5 EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSL SLSPGK CD8α transmembrane domain SEQ ID NO.6 IYIWAPLAGTCGVLLLSLVITLYC 41BB transmembrane domain SEQ ID NO. 7IISFFLALTSTALLFLLFFLTLRFSVV 41BB intracellular domain SEQ ID NO. 8KRGRKKLLYIFKQPFMRPVQTTQEEDGC SCRFPEEEEGGCEL CD3ζ intracellular domainSEQ ID NO. 9 RVKFSRSADAPAYQQGQNQLYNELNLG RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDALHMQA LPPR Linker SEQID NO. 10 GGGGSGGGGSGGGGS

TABLE 2 Sequence of the antibody fragments from Klon 43 used in theanti-CD123 scfv of the invention for the CD123 CAR of the invention ScFvSEQ ID sequences # amino acid sequence Klon43 SEQ IDMADYKDIVMTQSHKFMSTSVGDRVNITCKA light chain NO. 11SQNVDSAVAWYQQKPGQSPKALIYSASYRY variable SGVPDRFTGRGSGTDFTLTISSVQAEDLAVYYregion CQQYYSTPWTFGGGTKLEIKR Klon43 SEQ IDEVKLVESGGGLVQPGGSLSLSCAASGFTFTD heavy chain NO. 12YYMSWVRQPPGKALEWLALIRSKADGYTTE variable YSASVKGRFTLSRDDSQSILYLQMNALRPEDSregion ATYYCARDAAYYSYYSPEGAMDYWGQGTS VTVSS

TABLE 3 Sequence of the humanized antibody fragments from Klon 43 usedin the anti-CD123 scfv of the invention for the CD123 CAR of theinvention Functional domains SEQ ID # Raw amino acid sequence HumanizedscFv Klon43 SEQ ID NO. 18 MADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDS VariantVL1 AVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEI KR Humanized scFv Klon43 SEQ IDNO. 19 MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDS Variant VL2AVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEI KR Humanized scFv Klon43 SEQ IDNO. 20 MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDS Variant VL3AVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEI KR Humanized scFv Klon43 SEQ IDNO. 21 MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDS Variant VL4AVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEI KR Humanized scFv Klon43 SEQ IDNO. 22 MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDS Variant VL5AVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEI KR Humanized scFv Klon43 SEQ IDNO. 23 MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDS Variant VL6AVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEI KR Humanized scFv Klon43 SEQ IDNO. 24 EVKLVESGGGLVQPGRSLRLSCTASGFTFTDY Variant VH1YMSWVRQAPGKGLEWVGLIRSKADGYTTEYSAS VKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS Humanized scFv Klon43 SEQ ID NO. 25EVQLVESGGGLVQPGRSLRLSCTASGFTFTDY Variant VH2YMSWVRQAPGKGLEWVGLIRSKADGYTTEYSA SVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS Humanized scFv Klon43 SEQ ID NO. 26EVQLVESGGGLVQPGRSLRLSCTASGFTFTDY Variant VH3YMSWVRQAPGKGLEWVGLIRSKADGYTTEYSAS VKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS Humanized scFv Klon43 SEQ ID NO. 27EVQLVESGGGLVQPGRSLRLSCTASGFTFTDY Variant VH4YMSWVRQAPGKGLEWVGFIRSKADGYTTEYSAS VKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS Humanized scFv Klon43 SEQ ID NO. 28EVQLVESGGGLVQPGRSLRLSCTASGFTFTDY Variant VH5YMSWVRQAPGKGLEWVGFIRSKADGYTTEYAAS VKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS Humanized scFv Klon43 SEQ ID NO. 29EVQLVESGGGLVQPGRSLRLSCTASGFTFTDY Variant VH6YMSWVRQAPGKGLEWVGLIRSKADGYTTEYAA SVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS Humanized scFv Klon43 SEQ ID NO. 30EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSW Variant VH7VRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPE GAMDYWGQGTLVTVSS

TABLE 4 CAR of structure V-1 CAR Desig- CAR Structure nation signalFcγRIIIα CD8α 41BB- CD3ζ V-1 peptide VH VL hinge TM IC CD Klo43-1 SEQSEQ SEQ SEQ SEQ SEQ SEQ (SEQ ID ID ID ID ID ID ID ID NO: 31) NO. 1 NO.12 NO. 11 NO. 3 NO. 6 NO. 8 NO. 9

TABLE 5 CAR of structure V-3 CAR CAR Structure Designation signal CD8αCD8α 41BB- CD3ζ V-3 peptide VH VL hinge TM IC CD Klo43-3 SEQ SEQ SEQ SEQSEQ SEQ SEQ (SEQ ID ID ID ID ID ID ID ID NO: 32) NO. 1 NO. 12 NO. 11 NO.4 NO. 6 NO. 8 NO. 9

TABLE 6 CAR of structure V-5 CAR CAR Structure Designation signal IgG1CD8α 41BB- CD3ζ V-5 peptide VH VL hinge TM IC CD Klo43-5 SEQ SEQ SEQ SEQSEQ SEQ SEQ (SEQ ID ID ID ID ID ID ID ID NO: 33) NO. 1 NO. 12 NO. 11 NO.5 NO. 6 NO. 8 NO. 9

TABLE 7 Examples of mAb-specific epitopes also called mimotope (andtheir corresponding mAbs) that can be used in the extracellular domainof the CAR of the invention such as for example mimotopes and epitopewith their corresponding mAb Rituximab Mimotope SEQ ID NO 161 CPYSNPSLCPalivizumab Epitope SEQ ID NO 162 NSELLSLINDMPITNDQKKLMSNN CetuximabMimotope 1 SEQ ID NO 163 CQFDLSTRRLKC Mimotope 2 SEQ ID NO 164CQYNLSSRALKC Mimotope 3 SEQ ID NO 165 CVWQRWQKSYVC Mimotope 4 SEQ ID NO166 CMWDRFSRWYKC Nivolumab Epitope 1 SEQ ID NO 167SFVLNWYRMSPSNQTDKLAAFPEDR Epitope 2 SEQ ID NO 168 SGTYLCGAISLAPKAQIKEQBEND-10 Epitope SEQ ID NO 169 ELPTQGTFSNVSTNVSPAKPTTTA AlemtuzumabEpitope SEQ ID NO 170 GQNDTSQTSSPS

Sequences of the anti-CD123 CAR of the invention (SEQ ID NO.:31-160),preferred sequences are SEQ ID NO.:31, 32, 160 and 34-117, among 34-117more preferred are 76 to 117, even more preferred SEQ ID NO. 32, SEQ IDNO. 89, SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides:

-   -   1. A CD123 specific chimeric antigen receptor (CD123 CAR)        comprising        -   an extracellular domain comprising an extra cellular ligand            binding-domain comprising successively, a VH optionally            humanized a linker, preferably a linker of sequence (GGGGS)n            with n=1-4, preferably n=3, and a VL optionally humanized a            hinge,        -   a transmembrane domain and        -   a cytoplasmic domain.        -   at least one epitope specific for a monoclonal antibody            (mimotope).    -   2. the CD123 CAR according to 1 comprising        -   an extracellular domain comprising an extra cellular ligand            binding-domain comprising successively, a VH selected from            SEQ ID NO 12, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ            ID NO 27, SEQ ID NO 28, SEQ ID NO 29 and SEQ ID NO 30,            optionally humanized a linker, preferably a linker of            sequence (GGGGS)n with n=1-4, preferably n=3, and a VL            selected from SEQ ID NO 11, SEQ ID NO 18, SEQ ID NO 19, SEQ            ID NO 20, SEQ ID NO 21, SEQ ID NO 22 and SEQ ID NO 23,            optionally humanized a hinge,        -   a transmembrane domain from CD8 alpha, and        -   a cytoplasmic domain including a CD3 zeta signaling domain            and a co-stimulatory domain from 4-1BB.    -   3. The CD123 CAR according to 1 or 2 comprising no sequence        having identity the human CD28 NP_006130.1.    -   4. The CD123 CAR according to any one of 1 to 3 comprising a        sequence selected from SEQ ID NO 172, SEQ ID NO 173, SEQ ID NO        174, SEQ ID NO 175, SEQ ID NO 176, SEQ ID NO 177, SEQ ID NO 178,        SEQ ID NO 179, SEQ ID NO 180, SEQ ID NO 181, SEQ ID NO 182, SEQ        ID NO 183, SEQ ID NO 184, SEQ ID NO 185, SEQ ID NO 186 and SEQ        ID NO 187, optionally further comprising at least one SEQ ID No        161.    -   5. the CD123 CAR according to any one of 1 to 4 wherein said        extracellular domain comprises at least one epitope specific for        a monoclonal antibody (mimotope), selected from the list        consisting of SEQ ID NO 161, SEQ ID NO 162, SEQ ID NO 163, SEQ        ID NO 164, SEQ ID NO 165, SEQ ID NO 166, SEQ ID NO 167, SEQ ID        NO 168, SEQ ID NO 169 and SEQ ID NO 170, preferably of SEQ ID NO        161 and of SEQ ID NO 169.    -   6. the CD123 CAR according to any one of 1 to 5 comprising a        sequence selected from SEQ ID NO 160, SEQ ID NO 171, SEQ ID NO        188, SEQ ID NO 189, SEQ ID NO 190, SEQ ID NO 191, SEQ ID NO 192,        SEQ ID NO 193, SEQ ID NO 194, SEQ ID NO 195, SEQ ID NO 196, and        SEQ ID NO 197.    -   7. A polynucleotide encoding a CD123 specific chimeric antigen        receptor (CD123 CAR) according to any one of claims 1 to 6.    -   8. An expression vector comprising a polynucleotide according to        claim 7.    -   9. An expression vector comprising a backbone and at least one        sequence coding any one of the CD123 CAR defined in any one of        claims 1 to 6.    -   10. An expression vector comprising a backbone, an EF1 promotor,        an RQR8 open reading frame (RQR8 ORF), a sequence coding any one        of the CD123 CAR of 1 to 6.    -   11. A T Cell Receptor (TCR) knock-out (KO) or TCR and human        deoxycytidine kinase (dCK) KO engineered immune cell expressing        at the cell surface membrane a CD123 CAR according to any one of        1 to 6.    -   12. A TCR KO or TCR and dCK KO engineered immune cell comprising        a polynucleotide coding a CD123 specific chimeric antigen        receptor (CD123 CAR) according to any one of 1 to 6.    -   13. A TCR KO or TCR KO and dCK KO CD123 CAR-expressing        engineered immune cell according to 4 further expressing a        suicide domain at the cell surface.    -   14. A TCR KO or TCR KO and dCK KO CD123 CAR-expressing        engineered immune cell according to any one of the 11 to 13        wherein the expression of at least one MHC protein, is        suppressed.    -   15. A TCR KO or TCR KO and dCK KO CD123 CAR-expressing        engineered immune cell according to any one of 11 to 14 for use        in therapy.    -   16. A TCR KO or TCR KO and dCK KO CD123 CAR-expressing        engineered immune cell according to 15 wherein the condition is        acute myelogenous leukemia (AML), preferably refractory/relapsed        AML, BPDNL, or for use during or before bone marrow transplant.    -   17. A TCR KO or TCR KO and dCK KO CD123 CAR-expressing        engineered immune cell according to 15, for use as a treatment,        preferably as a treatment for a lymphoproliferative disorder,        more preferably for leukemia of lymphoma or for a treatment        selected from the group consisting of acute myelogenous        leukemia, chronic myelogenous leukemia, myelodysplastic        syndrome, acute lymphoid leukemia, chronic lymphoid leukemia,        and myelodysplastic syndrome and BPDNL.    -   18. A TCR KO or TCR KO and dCK KO CD123 CAR-expressing        engineered immune cell according to 15, for use as a treatment        for AML.

The present invention also provides:

1a. A polypeptide encoding chimeric antigen receptor (CAR) specific forCD123 comprising at least one extracellular binding domain, saidextracellular domain comprising at least one scFv said scfv iscomprising at least a VH chain and a VL chain binding specifically toCD123, wherein said extracellular binding domain comprises at least onemAb-specific epitope or mimotope.2a. The polypeptide according to 1a, wherein said mAb-specific epitopeis located between the VH and VL chains, or before the VH and the VL.

In one embodiment, the polypeptide encoding a chimeric antigen receptor(CAR) specific for CD123 is comprising a transmembrane domain (TM) and ahinge and said mAb-specific epitope is located between the scfv and thehinge.

The present invention provides:

3a. a polypeptide according to 1a or 2a, wherein said VH and VL chains,and mAb specific-epitope are bound together by at least one linker andby a hinge to the transmembrane domain of said CAR.4a. The polypeptide according to 3a, wherein the mAb-epitope is joinedto the VH and VL chains by two linkers.5a. The polypeptide according to any one of 1a to 3a wherein themAb-specific epitope is an epitope to be bound by an epitope-specificmAb for in vitro cell sorting and/or in vivo cell depletion of T cellsexpressing a CAR comprising such epitope.6a. The polypeptide according to any one of 1a to 5a, wherein thepolypeptide comprises one extracellular binding domain, a transmembranedomain, and an intracellular domain, wherein said extracellular bindingdomain comprises at least one mAb-specific epitope.7a. The polypeptide according to any one of 1a to 6a, wherein theextracellular binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10mAb-specific epitopes.8a. The polypeptide according to any one of 1a to 7a, wherein theextracellular binding domain comprises 1, 2, 3 or, 4 mAb-specificepitopes.9a. The polypeptide according to any one of 1a to 8a, wherein theextracellular binding domain comprises 2, 3 or, 4 mAb-specific epitopes.10a. The polypeptide according to any one of 1a to 9a, wherein theextracellular binding domain comprises the following sequenceV₁-L₁-V₂-(L)_(x)-Epitope1-(L)_(x)-;V₁-L₁-V₂-(L)_(x)-Epitope1-(L)_(x)-Epitope2-(L)_(x)-;V₁-L₁-V₂-(L)_(x)-Epitope1-(L)_(x)-Epitope2-(L)_(x)-Epitope3-(L)_(x)-;(L)_(x)-Epitope1-(L)_(x)-V₁-L₁-V₂;(L)_(x)-Epitope1-(L)_(x)-Epitope2-(L)_(x)-V₁-L₁-V₂;Epitope1-(L)_(x)-Epitope2-(L)_(x)-Epitope3-(L)_(x)-V₁-L₁-V₂;(L)_(x)-Epitope1-(L)_(x)-V₁-L₁-V₂-(L)_(x)-Epitope2-(L)_(x);(L)_(x)-Epitope1-(L)_(x)-V₁-L₁-V₂-(L)_(x)-Epitope2-(L)_(x)-Epitope3-(L)_(x)-;(L)_(x)-Epitope1-(L)_(x)-V₁-L₁-V₂-(L)_(x)-Epitope2-(L)_(x)-Epitope3-(L)_(x)-Epitope4-(L)_(x)-;(L)_(x)-Epitope1-(L)_(x)-Epitope2-(L)_(x)-V₁-L₁-V₂-(L)_(x)-Epitope3-(L)_(x)-;(L)_(x)-Epitope1-(L)_(x)-Epitope2-(L)_(x)-V₁-L₁-V₂-(L)_(x)-Epitope3-(L)_(x)-Epitope4-(L)_(x)-;V₁-(L)_(x)-Epitope1-(L)_(x)-V₂;V₁-(L)_(x)-Epitope1-(L)_(x)-V₂-(L)_(x)-Epitope2-(L)_(x);V₁-(L)_(x)-Epitope1-(L)_(x)-V₂-(L)_(x)-Epitope2-(L)_(x)-Epitope3-(L)_(x);V₁-(L)_(x)-Epitope1-(L)_(x)-V₂-(L)_(x)-Epitope2-(L)_(x)-Epitope3-(L)_(x)-Epitope4-(L)_(x);(L)_(x)-Epitope1-(L)_(x)-V₁-(L)_(x)-Epitope2-(L)_(x)-V₂; or,(L)_(x)-Epitope1-(L)_(x)-V₁-(L)_(x)-Epitope2-(L)_(x)-V₂-(L)_(x)-Epitope3-(L)_(x);wherein,V₁ is V_(L) and V₂ is V_(H) or V₁ is V_(H) and V₂ is V_(L);L₁ is a linker suitable to link the V_(H) chain to the V_(L) chain;L is a linker comprising glycine and serine residues, and eachoccurrence of L in the extracellular binding domain can be identical ordifferent to other occurrence of L in the same extracellular bindingdomain, and,x is 0 or 1 and each occurrence of x is selected independently from theothers; and,Epitope 1, Epitope 2 and Epitope 3 are mAb-specific epitopes and can beidentical or different.11a. The polypeptide according to 10a, wherein the extracellular bindingdomain comprises the following sequenceV₁-L₁-V₂-L-Epitope1; V₁-L₁-V₂-L-Epitope1-L;V₁-L₁-V₂-L-Epitope1-L-Epitope2; V₁-L₁-V₂-L-Epitope1-L-Epitope2-L;V₁-L₁-V₂-L-Epitope1-L-Epitope2-L-Epitope3;V₁-L₁-V₂-L-Epitope1-L-Epitope2-L-Epitope3-L; V₁-L₁-V₂-Epitope1;V₁-L₁-V₂-Epitope1-L; V₁-L₁-V₂-Epitope1-L-Epitope2;V₁-L₁-V₂-Epitope1-L-Epitope2-L; V₁-L₁-V₂-Epitope1-L-Epitope2-L-Epitope3;V₁-L₁-V₂-Epitope1-L-Epitope2-L-Epitope3-L; Epitope1-V₁-L₁-V₂;Epitope1-L-V₁-L₁-V₂; L-Epitope1-V₁-L₁-V₂; L-Epitope1-L-V₁-L₁-V₂;Epitope1-L-Epitope2-V₁-L₁-V₂; Epitope1-L-Epitope2-L-V₁-L₁-V₂;L-Epitope1-L-Epitope2-V₁-L₁-V₂; L-Epitope1-L-Epitope2-L-V₁-L₁-V₂;Epitope1-L-Epitope2-L-Epitope3-V₁-L₁-V₂;Epitope1-L-Epitope2-L-Epitope3-L-V₁-L₁-V₂;L-Epitope1-L-Epitope2-L-Epitope3-V₁-L₁-V₂;L-Epitope1-L-Epitope2-L-Epitope3-L-V₁-L₁-V₂; V₁-L-Epitope1-L-V₂;L-Epitope1-L-V₁-L-Epitope2-L-V₂; V₁-L-Epitope1-L-V₂-L-Epitope2-L;V₁-L-Epitope1-L-V₂-L-Epitope2-L-Epitope3;V₁-L-Epitope1-L-V₂-L-Epitope2-Epitope3;V₁-L-Epitope1-L-V₂-L-Epitope2-L-Epitope3-Epitope4;L-Epitope1-L-V₁-L-Epitope2-L-V₂-L-Epitope3-L;Epitope1-L-V₁-L-Epitope2-L-V₂-L-Epitope3-L;L-Epitope1-L-V₁-L-Epitope2-L-V₂-L-Epitope3;L-Epitope1-L-V₁-L₁-V₂-L-Epitope2-L;L-Epitope1-L-V₁-L₁-V₂-L-Epitope2-L-Epitope3;L-Epitope1-L-V₁-L₁-V₂-L-Epitope2-Epitope3, orEpitope1-L-V₁-L₁-V₂-L-Epitope2-L-Epitope3-Epitope4 whereinV₁ is V_(L) and V₂ is V_(H) or V₁ is V_(H) and V₂ is V_(L);L₁ is any linker suitable to link the V_(H) chain to the V_(L) chain;L is a linker comprising glycine and serine residues, and eachoccurrence of L in the extracellular binding domain can be identical ordifferent to other occurrences of L in the same extracellular bindingdomain, and,Epitope 1, Epitope 2 and Epitope 3 are mAb-specific epitopes and can beidentical or different.12a. The polypeptide according to 10a, wherein L₁ is a linker comprisingGlycine and/or Serine.13a. The polypeptide according to 12a, wherein L₁ is a linker comprisingthe amino acid sequence (Gly-Gly-Gly-Ser)_(n) or(Gly-Gly-Gly-Gly-Ser)_(n), where n is 1, 2, 3, 4 or 5 or a linkercomprising the amino acid sequence (Gly₄Ser)₄ or (Gly₄Ser)₃.14a. The polypeptide according to any one of 10a to 13a wherein L is alinker comprising Glycine and/or Serine.15a. The polypeptide according to 14a wherein L is a linker having anamino acid sequence selected from SGG, GGS, SGGS, SSGGS, GGGG, SGGGG,GGGGS, SGGGGS, GGGGGS, SGGGGGS, SGGGGG, GSGGGGS, GGGGGGGS, SGGGGGGG,SGGGGGGGS, or SGGGGSGGGGS.16a. The polypeptide according to 14a wherein L is a SGGGG, GGGGS orSGGGGS.17a. The polypeptide according to any one of 10a to 16a wherein Epitope1, Epitope 2, Epitope 3 and Epitope 4 are independently selected frommAb-specific epitopes specifically recognized by ibritumomab, tiuxetan,muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin,cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumabpegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab,omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab,vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab,ipilimumab, ofatumumab, panitumumab, QBEND-10 and ustekinumab.

In a preferred embodiment said Epitope 1, Epitope 2, are specificallyrecognized by rituximab and epitope 3 is specifically recognized byQBEND-10.

18a. The polypeptide according to any one of 10a to 16a wherein Epitope1, Epitope 2, Epitope 3 and Epitope 4 are independently selected frommAb-specific epitopes having an amino acid sequence of SEQ ID NO 161 to170.19a. The polypeptide according to any one of 10a to 18a wherein Epitope1 is a mAb-specific epitope having an amino acid sequence of SEQ ID NO161.20a. The polypeptide according to any one of claims 10a to 19a whereinEpitope 2 is a mAb-specific epitope having an amino acid sequence of SEQID NO 161.21a. The polypeptide according to any one of claims 10a to 20a whereinEpitope 3 is a mAb-specific epitope having an amino acid sequence of SEQID NO 161 or SEQ ID NO 169.22a. The polypeptide according to any one of claims 10a to 21a whereinEpitope 4 is a mAb-specific epitope having an amino acid sequence of SEQID NO 161.23a. The polypeptide according to claim 22a wherein Epitope 1, Epitope 2and Epitope 4 are a mAb-specific epitope having an amino acid sequenceof SEQ ID NO 161 and Epitope 3 is a mAb-specific epitope having an aminoacid sequence of SEQ ID NO 169.24a. The polypeptide according to anyone of 1a to 9a, wherein themAb-specific epitope is from one polypeptide selected from those listedin Table 7.25a. The polypeptide according to any one of 1a to 9a wherein themAb-specific epitope is selected from mAb-specific epitopes specificallyrecognized by ibritumomab, tiuxetan, muromonab-CD3, tositumomab,abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab,rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab,eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab,palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab,adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab,ofatumumab, panitumumab, QBEND-10 and ustekinumab.26a. The polypeptide according to any one of 1a to 9a wherein themAb-specific epitope is selected from mAb-specific epitope having anamino acid sequence of SEQ ID NO 161, SEQ ID NO 162, SEQ ID NO 163, SEQID NO 164, SEQ ID NO 165, SEQ ID NO 166, SEQ ID NO 167, SEQ ID NO 168,SEQ ID NO 169 or SEQ ID NO 170.27a. The polypeptide according to any one of 1a to 9a wherein themAb-specific epitope is has an amino acid sequence of SEQ ID NO 160.28a. The polypeptide according to anyone of 1a to 27a, wherein said VHand VL chains have an antigenic target sequence of over 80% identity,preferably over 90%, and more preferably over 95% with SEQ ID NO 11(CD123 antigen VH), SEQ ID NO 12 (CD123 antigen VL).29a. The polypeptide according to any one of 1a to 27a wherein saidCD123 antigen is a cell surface marker antigen.30a. The polypeptide according to any one of 1a to 27a wherein saidCD123 antigen is a tumor-associated surface antigen.31a. The polypeptide according to any one of 1a to 27a wherein saidantigen is CD123,32a. The polypeptide according to any one of 1a to 27a wherein VH and VLare selected from a VH of SEQ ID NO 11, SEQ ID NO 24 to SEQ ID NO 30 anda VL of SEQ ID NO 12; SEQ ID NO 18 to SEQ ID NO 23.33a. The polypeptide according to any one of 2a to 32a wherein the hingecomprises a CD8alpha hinge or a FcγRIII alpha hinge.34a. The polypeptide according to any one of 2a to 3a3 wherein thetransmembrane domain comprises the transmembrane region(s) CD8,35a. The polypeptide according to any one of 2a to 33a wherein thetransmembrane domain comprises the transmembrane region(s) of CD8 alpha.36a. The polypeptide according to any one of 2a to 33a wherein thetransmembrane domain comprises the transmembrane region(s) of CD8 alphaand a hinge from CD8 alpha.37a. The polypeptide according to any one of 2a to 37a wherein theintracellular domain comprises a CD3zeta signaling domain.38a. The polypeptide according to any one of 2a to 37a wherein theintracellular domain comprises a 4-1BB domain.39a. A polypeptide according to anyone of 1a to 38a, wherein the CAR isa single-chain CAR.40a. A polypeptide according to anyone of 1a to 38a wherein the CAR is amulti-chain CAR.40a bis A polypeptide according to anyone of 1a to 39a wherein the CAR asequence selected from SEQ ID NO 189 to SEQ ID NO 197.41a. A polynucleotide encoding a polypeptide according to anyone of 1ato 40a.42a. A polynucleotide encoding a chimeric antigen receptor according toanyone of 1a to 40a, wherein said CAR comprises a CD3 zeta signalingdomain and co-stimulatory domain from 4-113B.43a. An expression vector comprising a nucleic acid of 41a or 42a.44a. An engineered immune cell expressing at its cell surface apolypeptide according to anyone of 1a to 40a.45a. The engineered immune cell according to 44a, wherein said cell isderived from inflammatory T-lymphocytes, cytotoxic T-lymphocytes (CTL),regulatory T-lymphocytes or helper T-lymphocytes, preferably a CTL cell.46a. The engineered immune cell according to 44a or 45a for use as amedicament.47a. A method for engineering an immune cell of anyone of 44a-46a,comprising:(a) Providing an immune cell;(b) Introducing into said cell at least one polynucleotide encoding thechimeric antigen receptor according to anyone of 1a-40a.(c) Expressing said polynucleotide into said cell.48a. The method for engineering an immune cell of 47a, wherein immunecell is a T-cell.49a. A method for in vitro sorting engineered immune cell expressing atits cell surface a polypeptide comprising at least one mAb-specificepitope according to anyone of claims 1a to 40a comprising

-   -   contacting a population of immune cells comprising said        engineered immune cells with a monoclonal antibody specific for        the mAb-specific epitope;    -   selecting the cells that bind to the monoclonal antibody to        obtain a population of cells enriched in engineered immune cell.        50a. The method according to 49a wherein the monoclonal antibody        specific for the mAb-specific epitope is conjugated to a        fluorophore and the step of selecting the cells that bind to the        monoclonal antibody is done by Fluorescence Activated Cell        Sorting (FACS).        51a. The method according to 49a wherein the monoclonal antibody        specific for the mAb-specific epitope is conjugated to a        magnetic particle and the step of selecting the cells that bind        to the monoclonal antibody is done by Magnetic Activated Cell        Sorting (MACS).        52a. The method according to any one of 49 to 51 wherein the        polypeptide comprises an mAb-specific epitope having an amino        acid sequence of SEQ ID NO 160 and the monoclonal antibody is        rituximab.        53a. The method according to any one of 49a to 51a wherein the        polypeptide comprises an mAb-specific epitope having an amino        acid sequence of SEQ ID NO 169 and the antibody used to contact        the population of immune cells is QBEND-10.        54a. The method according to any one of 49a to 53a wherein the        population of cells enriched in engineered immune cell comprises        at least 70%, 75%, 80%, 85%, 90%, 95% of CAR-expressing immune        cells.        55a. A method for in vivo depleting an engineered immune cell        expressing at its cell surface a polypeptide comprising at least        one mAb-specific epitope according to anyone of 1a to 40a in a        patient, comprising contacting said engineered immune cell with        at least one epitope-specific mAb.        56a. The method according to 56a wherein the mAb-specific        epitope is a CD20 epitope or mimotope and the epitope-specific        mAb is rituximab.        57a. The method according to 57a wherein the mAb-specific        epitope has an amino acid sequence of SEQ ID NO 160.        58a. The method according to any one of 56a to 58a wherein the        epitope-specific mAb is conjugated with a molecule able to        activate the complement system.        59a. The method according to any one of 56a to 58a wherein,        wherein a cytotoxic drug is coupled to the epitope-specific mAb.        60a. A method for in vivo depleting an engineered immune cell        expressing at its cell surface a polypeptide comprising at least        one mAb-specific epitope according to anyone of 1a to 40a in a        patient, comprising contacting said engineered immune cell with        bi-specific mAb (BsAb) able to bind both the mAb-specific        epitope borne on said cells and to an surface antigen borne on        an effector (and cytotoxic) cell.        61a. A method according to any one of 47a to 60a, wherein said        immune cell is a T-cell.

specifically, the present invention also provides 1b a CD123 specificchimeric antigen receptor (CD123 CAR) comprising

-   -   an extracellular domain comprising an extra cellular ligand        binding-domain comprising successively, a VH selected from SEQ        ID NO 12, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO        27, SEQ ID NO 28, SEQ ID NO 29 and SEQ ID NO 30, optionally        humanized at least one linker, preferably a linker of sequence        (GGGGS)n with n=1-4, more preferably n=3, and a VL selected from        SEQ ID NO 11, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID        NO 21, SEQ ID NO 22 and SEQ ID NO 23, optionally humanized a        hinge,    -   a transmembrane domain from CD8 alpha, and    -   a cytoplasmic domain including a CD3 zeta signaling domain and a        co-stimulatory domain from 4-1BB.

A CD123 specific chimeric antigen receptor (CD123 CAR) comprising

-   -   an extracellular domain comprising an extra cellular ligand        binding-domain comprising successively, a VH selected from SEQ        ID NO 12, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO        27, SEQ ID NO 28, SEQ ID NO 29 and SEQ ID NO 30, a linker of SEQ        ID NO. 10, and a VL selected from SEQ ID NO 11, SEQ ID NO 18,        SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22 and SEQ        ID NO 23, a hinge,    -   a transmembrane domain from CD8 alpha, and    -   a cytoplasmic domain including a CD3 zeta signaling domain and a        co-stimulatory domain from 4-1BB.        2b the present invention provides a CD123 CAR according to 1b        comprising no sequence having identity the human CD28        NP_006130.1.        3b The present invention provides a CD123 CAR according to 1b or        2b wherein said extracellular domain comprises at least one        epitope specific for a monoclonal antibody of sequence selected        from SEQ ID NO 161 to SEQ ID NO 170, or a combination thereof,        preferably at least one epitope specific for a monoclonal        antibody of SEQ ID NO 161.

The present invention provides a CD123 CAR wherein the CD123 CARcomprises a sequence selected from SEQ ID NO 189 to SEQ ID NO 197.

The present invention provides a CD123 CAR wherein the CD123 CARcomprises a sequence of SEQ ID NO 190

The present invention provides a CD123 CAR wherein the CD123 CARcomprises a sequence of SEQ ID NO 191

The present invention provides a CD123 CAR wherein the CD123 CARcomprises a sequence of SEQ ID NO 192

The present invention provides a CD123 CAR wherein the CD123 CARcomprises a sequence of SEQ ID NO 193

The present invention provides a CD123 CAR wherein the CD123 CARcomprises a sequence of SEQ ID NO 194

The present invention provides a CD123 CAR wherein the CD123 CARcomprises a sequence of SEQ ID NO 195

The present invention provides a CD123 CAR wherein the CD123 CARcomprises a sequence of SEQ ID NO 196

The present invention provides a CD123 CAR wherein the CD123 CARcomprises a sequence of SEQ ID NO 197.

The present invention provides a CD123 CAR according to 1b or 2b whereinsaid extracellular domain comprises an scfv comprising at least oneepitope specific for a monoclonal antibody of sequence selected from SEQID NO 161, SEQ ID NO 162, SEQ ID NO 163, SEQ ID NO 164, SEQ ID NO 165,SEQ ID NO 166, SEQ ID NO 167, SEQ ID NO 168 and SEQ ID NO 169, and SEQID NO 170.

Preferably, the present invention provides a CD123 CAR according to 1bor 2b wherein said extracellular domain comprises an scfv comprising twoepitopes specific for a monoclonal antibody recognizing SEQ ID NO 161and one other two epitopes specific for a monoclonal antibodyrecognizing SEQ ID NO 169.

4b. The present invention provides a CD123 CAR according to 3bcomprising a sequence of SEQ ID NO 171.5b The present invention provides a CD123 specific chimeric antigenreceptor (CD123 CAR) according to 1b having a sequence selected from SEQID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33 and SEQ ID No 34 to SEQ ID No160.6b The present invention provides a CD123 specific chimeric antigenreceptor (CD123 CAR) according to any one of 1b to 5b having a sequenceselected from SEQ ID No 34 to SEQ ID No 160.7b The present invention provides a polynucleotide encoding a CD123specific chimeric antigen receptor (CD123 CAR) according to any one of1b to 6b.8b The present invention provides an expression vector comprising apolynucleotide according to 7.9b) The present invention provides an expression vector comprising abackbone and a sequence coding any one of the CD123 CAR defined in anyone of 1b to 6b.10b) The present invention provides an expression vector comprising abackbone, preferably a backbone comprising an EF1 promotor, an RQR8 openreading frame (RQR8 ORF), a sequence coding any one of the CD123 CAR of1 to 8 embodiments above.11b) The present invention provides a T Cell Receptor (TCR) knock-out(KO) or TCR and human deoxycytidine kinase (dCK) KO engineered immunecell expressing at the cell surface membrane a CD123 CAR according toany one of 1b to 9b.12b) The present invention provides a TCR KO or TCR and dCK KOengineered immune cell comprising a polynucleotide coding a CD123specific chimeric antigen receptor (CD123 CAR) according to any one of1b to 6b.

A TCR KO or TCR and dCK KO engineered immune cell comprising anexpression vector comprising a polynucleotide encoding a CD123 specificchimeric antigen receptor (CD123 CAR) of the invention.

A TCR KO or TCR and dCK KO engineered immune cell comprising anexpression vector comprising a backbone and a sequence comprising apolynucleotide encoding a CD123 specific chimeric antigen receptor(CD123 CAR) of the invention.

A TCR KO or TCR and dCK KO engineered immune cell comprising anexpression vector comprising a backbone, an EF1 promotor, an RQR8 openreading frame (RQR8 ORF), a sequence coding any one of the CD123 CAR ofthe invention.

13b) The present invention provides a TCR KO or TCR KO and dCK KO CD123CAR-expressing engineered immune cell according to 11b or 12b furtherexpressing a suicide domain at the cell surface.(14b) The present invention provides a TCR KO or TCR KO and dCK KO CD123CAR-expressing engineered immune T cell according to any one of 10b to13b wherein said suicide domain at the cell surface is inserted into theCD123 CAR extracellular domain.

A TCR KO and dCK KO CD123 CAR-expressing engineered immune cellaccording to any one of 11b to 14b, wherein the dCK gene is deletedconferring resistance to purine nucleotide analogs (PNA).

(15b) The present invention provides a TCR KO or TCR KO and dCK KO CD123CAR—expressing engineered immune cell according to any one of the 11b to14b wherein expression of at least one MHC protein, is suppressed.16b) The present invention provides a TCR KO or TCR KO and dCK KO CD123CAR-expressing engineered immune cell according to any one of (11b) to(15b) for use in therapy.

The present invention provides a pharmaceutical composition comprising aCD123 CAR as in 1b) to 6b).

The present invention provides a pharmaceutical composition comprising aTCR KO or TCR KO and dCK KO CD123 CAR-expressing engineered immune cellaccording to any one of 11b to 15b.

17b) The present invention provides a TCR KO or TCR KO and dCK KO CD123CAR-expressing engineered immune cell according to 16b wherein thecondition is acute myelogenous leukemia (AML), preferablyrefractory/relapsed AML, BPDNL, or for use during bone marrowtransplant.18b) The present invention provides a TCR KO or TCR KO and dCK KO CD123CAR-expressing engineered immune cell according to 16b, for use as atreatment, preferably as a treatment for a lymphoproliferative disorder,more preferably for leukemia of lymphoma or for a treatment selectedfrom the group consisting of acute myelogenous leukemia, chronicmyelogenous leukemia, myelodysplastic syndrome, acute lymphoid leukemia,chronic lymphoid leukemia, and myelodysplastic syndrome and BPDNL.

The present invention provides a pharmaceutical composition as above foruse in therapy.

The present invention provides a pharmaceutical composition as above foruse in therapy for the treatment of acute myelogenous leukemia (AML),preferably refractory/relapsed AML, BPDNL, or for use during bone marrowtransplant.

The present invention provides a pharmaceutical composition for use as atreatment, preferably as a treatment for a lymphoproliferative disorder,more preferably for leukemia of lymphoma or for a treatment selectedfrom the group consisting of acute myelogenous leukemia, chronicmyelogenous leukemia, myelodysplastic syndrome, acute lymphoid leukemia,chronic lymphoid leukemia, and myelodysplastic syndrome and BPDNL.

Unless specifically defined herein, all technical and scientific termsused have the same meaning as commonly understood by a skilled artisanin the fields of gene therapy, pharmacology, immunology, biochemistry,genetics, and molecular biology.

All methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,with suitable methods and materials being described herein. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willprevail. Further, the materials, methods, and examples are illustrativeonly and are not intended to be limiting, unless otherwise specified.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, CurrentProtocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley andson Inc, Library of Congress, USA); Molecular Cloning: A LaboratoryManual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, NewYork: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis(M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; NucleicAcid Hybridization (B. D. Harries & S. J. Higgins eds. 1984);Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984);Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987);Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A PracticalGuide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J.Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York),specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, “GeneExpression Technology” (D. Goeddel, ed.); Gene Transfer Vectors ForMammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold SpringHarbor Laboratory); Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986).

The present invention discloses an engineered immune cell (TCR KO and/ordck KO) expressing a CD123 specific chimeric antigen receptor (“CD123CAR” or “CAR”) comprising an extra cellular ligand binding-domaincomprising a VH and a VL from a monoclonal anti-CD123 antibody KLON43 orhumanized VH and humanized VL sequence thereof, a hinge from CD8 alphaof from FcγRIIIα, a transmembrane domain from CD8 alpha, a cytoplasmicdomain including a CD3 zeta signaling domain and a co-stimulatory domainfrom 4-1BB, said 123 CAR having sequence identity with either SEQ ID NO.31, SEQ ID NO. 32, or SEQ ID NO. 33, or any one of SEQ ID NO. 34 to SEQID NO. 117, SEQ ID NO. 160 or preferably, SEQ ID NO 188 to SEQ ID NO197.

Preferably the present invention discloses an engineered immune cell(TCR KO and/or dck KO) expressing a CD123 specific CAR of SEQ ID No 31,32 or 160, more preferably an engineered immune cell (TCR KO) expressinga CD123 specific CAR of SEQ ID No 31, and even more preferably anengineered immune cell (TCR KO and dck KO) expressing a CD123 specificCAR of SEQ ID No 32.

Advantageously said the present invention discloses an engineered immunecell (TCR KO and/or dck KO) expressing a CD123 specific CAR of SEQ ID No31, 32 or 160 and a suicide domain, more preferably an engineered immunecell (TCR KO) expressing a CD123 specific CAR of SEQ ID No 31 and asuicide domain, and even more preferably an engineered immune cell (TCRKO and dck KO) expressing a CD123 specific CAR of SEQ ID No 32, and evenmore more preferably an engineered immune cell (TCR KO and dck KO)expressing a CD123 specific CAR of SEQ ID No 160, or of one of thefollowing sequences SEQ ID NO 188 to SEQ ID NO 197.

The present invention discloses an engineered immune cell (TCR KO and/ordck KO) expressing a CD123 specific CAR having one of the polypeptidestructure selected from V1, V3 as illustrated in FIG. 2, said structurecomprising an extra cellular ligand binding-domain comprising VH and VLfrom a monoclonal anti-CD123 antibody KLON 43, a hinge from CD8alpha orFcγRIIIα, a transmembrane domain from CD8 alpha, a cytoplasmic domainincluding a CD3 zeta signaling domain and a co-stimulatory domain from4-1BB, and no sequence from CD28 said 123 CAR having at least 80%sequence identity with either SEQ ID NO. 31, SEQ ID NO. 32, preferablySEQ ID NO. 32.

The present invention discloses an engineered immune cell (TCR KO and/ordck KO) expressing a CD123 specific CAR having one of the polypeptidestructure selected from V1, V3 as illustrated in FIG. 2, said structurecomprising an extra cellular ligand binding-domain comprising VH and VLfrom a monoclonal anti-CD123 antibody KLON 43, a hinge from CD8alpha orFcγRIIIα, a transmembrane domain from CD8 alpha, a cytoplasmic domainincluding a CD3 zeta signaling domain and a co-stimulatory domain from4-1BB, and no sequence from CD28 said 123 CAR having at least 80%sequence identity with either SEQ ID NO. 34 to SEQ ID NO. 159,preferably SEQ ID NO. 34 to SEQ ID NO. 117, preferably SEQ ID NO. 76 toSEQ ID NO. 117.

In one embodiment the present invention discloses an engineered immunecell (TCR KO and/or dck KO) expressing a CD123 specific CAR having asequence selected from SEQ ID NO. 34, SEQ ID NO. 76, SEQ ID NO. 36, SEQID NO. 78; SEQ ID NO. 37, SEQ ID NO. 79, SEQ ID NO. 41, SEQ ID NO. 83,SEQ ID NO. 42, SEQ ID NO. 8), SEQ ID NO. 43, SEQ ID NO. 85, SEQ ID NO.46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 88, SEQ IDNO. 89, SEQ ID NO. 90, SEQ ID NO. 91. SEQ ID NO. 52, SEQ ID NO. 53, SEQID NO. 54, SEQ ID NO. 55, SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96,SEQ ID NO. 97.

Preferably, the present invention discloses an engineered immune cell(TCR KO and/or dck KO) expressing a CD123 specific CAR having a sequenceselected from SEQ ID NO. 34, SEQ ID NO. 76, SEQ ID NO. 36, SEQ ID NO.78; SEQ ID NO. 43, SEQ ID NO. 85, SEQ ID NO. 46, SEQ ID NO. 47, SEQ IDNO. 48, SEQ ID NO. 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 52, SEQID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 94, SEQ ID NO. 95,SEQ ID NO. 96 SEQ ID NO. 97.

More preferably, the present invention discloses an engineered immunecell (TCR KO and/or dck KO) expressing a CD123 specific CAR having asequence selected from SEQ ID NO. 47, SEQ ID NO. 89, SEQ ID NO. 52, SEQID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 94, SEQ ID NO. 95,SEQ ID NO. 96, SEQ ID NO. 97.

And even more preferably the present invention discloses an engineeredimmune cell (TCR KO and/or dck KO) expressing a CD123 specific 123 CARhaving a sequence selected from SEQ ID NO. 32, SEQ ID NO. 89, SEQ ID NO.94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97.

The most preferred embodiment the present invention discloses anengineered immune cell (TCR KO and/or dck KO) expressing a CD123specific 123 CAR having a sequence of SEQ ID NO. 172 comprising at leastone epitope recognized by a specific monoclonal antibody selected fromSEQ ID NO. 161 to SEQ ID NO. 170, preferably two of SEQ ID NO 161 andone from SEQ ID NO 169.

Another most preferred embodiment discloses an engineered immune cell(TCR KO and/or dck KO) expressing a CD123 specific 123 CAR having asequence of SEQ ID NO. 173 comprising at least one epitope recognized bya specific monoclonal antibody selected from SEQ ID NO. 161 to SEQ IDNO. 170, preferably two of SEQ ID NO 161 and one from SEQ ID NO 169.

Another most preferred embodiment discloses an engineered immune cell(TCR KO and/or dck KO) expressing a CD123 specific 123 CAR having asequence of SEQ ID NO. 174 comprising at least one epitope recognized bya specific monoclonal antibody selected from SEQ ID NO. 161 to SEQ IDNO. 170, preferably two of SEQ ID NO 161 and one from SEQ ID NO 169.

Another most preferred embodiment discloses an engineered immune cell(TCR KO and/or dck KO) expressing a CD123 specific 123 CAR having asequence of SEQ ID NO. 175 comprising at least one epitope recognized bya specific monoclonal antibody selected from SEQ ID NO. 161 to SEQ IDNO. 170, preferably two of SEQ ID NO 161 and one from SEQ ID NO 169.

Another most preferred embodiment discloses an engineered immune cell(TCR KO and/or dck KO) expressing a CD123 specific 123 CAR having asequence of SEQ ID NO. 176 comprising at least one epitope recognized bya specific monoclonal antibody selected from SEQ ID NO. 161 to SEQ IDNO. 170, preferably two of SEQ ID NO 161 and one from SEQ ID NO 169.

Another most preferred embodiment discloses an engineered immune cell(TCR KO and/or dck KO) expressing a CD123 specific 123 CAR having asequence of SEQ ID NO. 177 comprising at least one epitope recognized bya specific monoclonal antibody selected from SEQ ID NO. 161 to SEQ IDNO. 170, preferably two of SEQ ID NO 161 and one from SEQ ID NO 169.

Another most preferred embodiment discloses an engineered immune cell(TCR KO and/or dck KO) expressing a CD123 specific 123 CAR having asequence of SEQ ID NO. 178 comprising at least one epitope recognized bya specific monoclonal antibody selected from SEQ ID NO. 161 to SEQ IDNO. 170, preferably two of SEQ ID NO 161 and one from SEQ ID NO 169.

Another most preferred embodiment discloses an engineered immune cell(TCR KO and/or dck KO) expressing a CD123 specific 123 CAR having asequence of SEQ ID NO. 179 comprising at least one epitope recognized bya specific monoclonal antibody selected from SEQ ID NO. 161 to SEQ IDNO. 170, preferably two of SEQ ID NO 161 and one from SEQ ID NO 169.

Another most preferred embodiment discloses an engineered immune cell(TCR KO and/or dck KO) expressing a CD123 specific 123 CAR having asequence of SEQ ID NO. 180 comprising at least one epitope recognized bya specific monoclonal antibody selected from SEQ ID NO. 161 to SEQ IDNO. 170, preferably two of SEQ ID NO 161 and one from SEQ ID NO 169.

Another most preferred embodiment discloses an engineered immune cell(TCR KO and/or dck KO) expressing a CD123 specific 123 CAR having asequence of SEQ ID NO. 181 comprising at least one epitope recognized bya specific monoclonal antibody selected from SEQ ID NO. 161 to SEQ IDNO. 170, preferably two of SEQ ID NO 161 and one from SEQ ID NO 169.

Another most preferred embodiment discloses an engineered immune cell(TCR KO and/or dck KO) expressing a CD123 specific 123 CAR having asequence of SEQ ID NO. 182 comprising at least one epitope recognized bya specific monoclonal antibody selected from SEQ ID NO. 161 to SEQ IDNO. 170, preferably two of SEQ ID NO 161 and one from SEQ ID NO 169.

Another most preferred embodiment discloses an engineered immune cell(TCR KO and/or dck KO) expressing a CD123 specific 123 CAR having asequence of SEQ ID NO. 183 comprising at least one epitope recognized bya specific monoclonal antibody selected from SEQ ID NO. 161 to SEQ IDNO. 170, preferably two of SEQ ID NO 161 and one from SEQ ID NO 169.

Another most preferred embodiment discloses an engineered immune cell(TCR KO and/or dck KO) expressing a CD123 specific 123 CAR having asequence of SEQ ID NO. 184 comprising at least one epitope recognized bya specific monoclonal antibody selected from SEQ ID NO. 161 to SEQ IDNO. 170, preferably two of SEQ ID NO 161 and one from SEQ ID NO 169.

Another most preferred embodiment discloses an engineered immune cell(TCR KO and/or dck KO) expressing a CD123 specific 123 CAR having asequence of SEQ ID NO. 185 comprising at least one epitope recognized bya specific monoclonal antibody selected from SEQ ID NO. 161 to SEQ IDNO. 170, preferably two of SEQ ID NO 161 and one from SEQ ID NO 169.

Another most preferred embodiment discloses an engineered immune cell(TCR KO and/or dck KO) expressing a CD123 specific 123 CAR having asequence of SEQ ID NO. 186 comprising at least one epitope recognized bya specific monoclonal antibody selected from SEQ ID NO. 161 to SEQ IDNO. 170, preferably two of SEQ ID NO 161 and one from SEQ ID NO 169.

Another most preferred embodiment discloses an engineered immune cell(TCR KO and/or dck KO) expressing a CD123 specific 123 CAR having asequence of SEQ ID NO. 187 comprising at least one epitope recognized bya specific monoclonal antibody selected from SEQ ID NO. 161 to SEQ IDNO. 170, preferably two of SEQ ID NO 161 and one from SEQ ID NO 169.

In another most preferred embodiment the present invention discloses anengineered immune cell (TCR KO and/or dck KO) expressing a CD123specific 123 CAR having a sequence of SEQ ID NO. 188

In another most preferred embodiment the present invention discloses anengineered immune cell (TCR KO and/or dck KO) expressing a CD123specific 123 CAR having a sequence of SEQ ID NO. 189.

In another most preferred embodiment the present invention discloses anengineered immune cell (TCR KO and/or dck KO) expressing a CD123specific 123 CAR having a sequence of SEQ ID NO. 190.

In another most preferred embodiment the present invention discloses anengineered immune cell (TCR KO and/or dck KO) expressing a CD123specific 123 CAR having a sequence of SEQ ID NO. 191.

In another most preferred embodiment the present invention discloses anengineered immune cell (TCR KO and/or dck KO) expressing a CD123specific 123 CAR having a sequence of SEQ ID NO. 192.

In another most preferred embodiment the present invention discloses anengineered immune cell (TCR KO and/or dck KO) expressing a CD123specific 123 CAR having a sequence of SEQ ID NO. 193.

In another most preferred embodiment the present invention discloses anengineered immune cell (TCR KO and/or dck KO) expressing a CD123specific 123 CAR having a sequence of SEQ ID NO. 194

In another most preferred embodiment the present invention discloses anengineered immune cell (TCR KO and/or dck KO) expressing a CD123specific 123 CAR having a sequence of SEQ ID NO. 195.

In another most preferred embodiment the present invention discloses anengineered immune cell (TCR KO and/or dck KO) expressing a CD123specific 123 CAR having a sequence of SEQ ID NO. 196.

In another most preferred embodiment the present invention discloses anengineered immune cell (TCR KO and/or dck KO) expressing a CD123specific 123 CAR having a sequence of SEQ ID NO. 197.

The present invention discloses an engineered immune cell (TCR KO and/ordck KO) expressing a CD123 specific 123 CAR which extracellular bindingdomain is modified in such a way to allow both cell sorting and celldepletion. This structure named “mAb-driven sorting/depletion system” or“epitope specific for a monoclonal antibody” or “mimotope” is a selectedepitope inserted within the extracellular domain of the anti-CD123 CARof the invention, in particular into the anti-CD123 scFv; or between theTM and the hinge; this epitope having a specificity to be recognized bya specific antibody (preferably mAb). Given the fact that mainly theexternal ligand binding domain of the CAR is modified to include theepitope, different CAR architectures can be envisioned: single-chain ormulti-chain. The chimeric scFv of the invention, which is formed of theVH and VL polypeptides and of the specific epitope(s) may itself havedifferent structures depending on the position of insertion of theepitope and the use of linkers. The present invention also relates tothe resulting method for sorting and/or depleting the engineered immunecells endowed with the modified CARs.

In some embodiments, the extracellular binding domain of the anti-CD123CAR comprises the following sequence (including mimotopes) (Nterm islocated on the left hand side):

V₁-L₁-V₂-(L)_(x)-Epitope1-(L)_(x);V₁-L₁-V₂-(L)_(x)-Epitope1-(L)_(x)-Epitope2-(L)_(x);V₁-L₁-V₂-(L)_(x)-Epitope1-(L)_(x)-Epitope2-(L)_(x)-Epitope3-(L)_(x);(L)_(x)-Epitope1-(L)_(x)-V₁-L₁-V₂;(L)_(x)-Epitope1-(L)_(x)-Epitope2-(L)_(x)-V₁-L₁-V₂;Epitope1-(L)_(x)-Epitope2-(L)_(x)-Epitope3-(L)_(x)-V₁-L₁-V₂;(L)_(x)-Epitope1-(L)_(x)-V₁-L₁-V₂-(L)_(x)-Epitope2-(L)_(x);(L)_(x)-Epitope1-(L)_(x)-V₁-L₁-V₂-(L)_(x)-Epitope2-(L)_(x)-Epitope3-(L)_(x);(L)_(x)-Epitope1-(L)_(x)-V₁-L₁-V₂-(L)_(x)-Epitope2-(L)_(x)-Epitope3-(L)_(x)-Epitope4-(L)_(x);(L)_(x)-Epitope1-(L)_(x)-Epitope2-(L)_(x)-V₁-L₁-V₂-(L)_(x)-Epitope3-(L)_(x);(L)_(x)-Epitope1-(L)_(x)-Epitope2-(L)_(x)-V₁-L₁-V₂-(L)_(x)-Epitope3-(L)_(x)-Epitope4-(L)_(x);V₁-(L)_(x)-Epitope1-(L)_(x)-V₂;V₁-(L)_(x)-Epitope1-(L)_(x)-V₂-(L)_(x)-Epitope2-(L)_(x);V₁-(L)_(x)-Epitope1-(L)_(x)-V₂-(L)_(x)-Epitope2-(L)_(x)-Epitope3-(L)_(x);V₁-(L)_(x)-Epitope1-(L)_(x)-V₂-(L)_(x)-Epitope2-(L)_(x)-Epitope3-(L)_(x)-Epitope4-(L)_(x);(L)_(x)-Epitope1-(L)_(x)-V₁-(L)_(x)-Epitope2-(L)_(x)-V₂;(L)_(x)-Epitope1-(L)_(x)-V₁-(L)_(x)-Epitope2-(L)_(x)-V₂-(L)_(x)-Epitope3-(L)_(x);V₁-L₁-V₂-L-Epitope1;V₁-L₁-V₂-L-Epitope1-L;V₁-L₁-V₂-L-Epitope1-L-Epitope2;V₁-L₁-V₂-L-Epitope1-L-Epitope2-L;V₁-L₁-V₂-L-Epitope1-L-Epitope2-L-Epitope3;V₁-L₁-V₂-L-Epitope1-L-Epitope2-L-Epitope3-L;V₁-L₁-V₂-Epitope1;V₁-L₁-V₂-Epitope1-L;V₁-L₁-V₂-Epitope1-L-Epitope2;V₁-L₁-V₂-Epitope1-L-Epitope2-L;V₁-L₁-V₂-Epitope1-L-Epitope2-L-Epitope3;V₁-L₁-V₂-Epitope1-L-Epitope2-L-Epitope3-L;Epitope1-V₁-L₁-V₂;Epitope1-L-V₁-L₁-V₂;L-Epitope1-V₁-L₁-V₂;L-Epitope1-L-V₁-L₁-V₂;Epitope1-L-Epitope2-V₁-L₁-V₂;Epitope1-L-Epitope2-L-V₁-L₁-V₂;L-Epitope1-L-Epitope2-V₁-L₁-V₂;L-Epitope1-L-Epitope2-L-V₁-L₁-V₂;Epitope1-L-Epitope2-L-Epitope3-V₁-L₁-V₂;Epitope1-L-Epitope2-L-Epitope3-L-V₁-L₁-V₂;L-Epitope1-L-Epitope2-L-Epitope3-V₁-L₁-V₂;L-Epitope1-L-Epitope2-L-Epitope3-L-V₁-L₁-V₂;V₁-L-Epitope1-L-V₂;L-Epitope1-L-V₁-L-Epitope2-L-V₂;V₁-L-Epitope1-L-V₂-L-Epitope2-L;V₁-L-Epitope1-L-V₂-L-Epitope2-L-Epitope3;V₁-L-Epitope1-L-V₂-L-Epitope2-Epitope3;V₁-L-Epitope1-L-V₂-L-Epitope2-L-Epitope3-Epitope4;L-Epitope1-L-V₁-L-Epitope2-L-V₂-L-Epitope3-L;Epitope1-L-V₁-L-Epitope2-L-V₂-L-Epitope3-L;L-Epitope1-L-V₁-L-Epitope2-L-V₂-L-Epitope3;L-Epitope1-L-V₁-L₁-V₂-L-Epitope2-L;L-Epitope1-L-V₁-L₁-V₂-L-Epitope2-L-Epitope3;L-Epitope1-L-V₁-L₁-V₂-L-Epitope2-Epitope3, or,Epitope1-L-V₁-L₁-V₂-L-Epitope2-L-Epitope3-Epitope4.wherein,V₁ and V₂ are V_(H) and V_(L) of an ScFv anti-CD123 (i.e, V₁ is V_(L)and V₂ is V_(H) or V₁ is V_(H) and V₂ is V_(L));L₁ is any linker suitable to link the VH chain to the VL chain in aScFv;L is a linker, preferably comprising glycine and serine residues, andeach occurrence of L in the extracellular binding domain can beidentical or different to other occurrence of L in the sameextracellular binding domain, and,x is 0 or 1 and each occurrence of x is independently from the others;and,epitope 1, epitope 2 and epitope 3 are mAb-specific epitopes (ormimotopes) and can be identical or different.

In some embodiments, the extracellular binding domain comprises thefollowing sequence (N-term is located on the left hand side):

V_(H)-L₁-V₁-L-Epitope1-L-Epitope2-L;L-Epitope1-L-V_(H)-L-Epitope2-L-V₁-L-Epitope3-L;V₁-L¹-V_(H)-L-Epitope1-L-Epitope2-L; or,L-Epitope1-L-V₁-L-Epitope2-L-V_(H)-L-Epitope3-L.wherein L, L1, epitope 1, epitope 2 and epitope 3 are as defined above.

L₁ is a linker comprising Glycine and/or Serine. In some embodiment, L₁is a linker comprising the amino acid sequence (Gly-Gly-Gly-Ser)_(n) or(Gly-Gly-Gly-Gly-Ser)_(n), where n is 1, 2, 3, 4 or 5. In someembodiments L₁ is (Gly₄Ser)₄ or (Gly₄Ser)₃.

L is a flexible linker, preferably comprising Glycine and/or Serine. Insome embodiments, L has an amino acid sequence selected from SGG, GGS,SGGS, SSGGS, GGGG, SGGGG, GGGGS, SGGGGS, GGGGGS, SGGGGGS, SGGGGG,GSGGGGS, GGGGGGGS, SGGGGGGG, SGGGGGGGS, or SGGGGSGGGGS preferably SGG,SGGS, SSGGS, GGGG, SGGGGS, SGGGGGS, SGGGGG, GSGGGGS or SGGGGSGGGGS. Insome embodiment, when the extracellular binding domain comprises severaloccurrences of L, all the Ls are identical. In some embodiments, whenthe extracellular binding domain comprises several occurrences of L, theLs are not all identical. In some embodiments, L is SGGGGS, In someembodiments, the extracellular binding domain comprises severaloccurrences of L and all the Ls are SGGGGS.

In some embodiments, Epitope 1, Epitope 2 and Epitope 3 are identical ordifferent and are selected from mAb-specific epitopes having an aminoacid sequence as in Table 7.

In a preferred embodiments, Epitope 1, Epitope 2 are identical ordifferent and are selected from mAb-specific epitopes specificallyrecognized by ibritumomab, tiuxetan, muromonab-CD3, tositumomab,abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab,rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab,eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab,palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab,adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab,ofatumumab, panitumumab, QBEND-10, alemtuzumab or ustekinumab,preferably those already approved for medical use, such as rituximab asa non-limiting example.

Finally, the invention encompasses therapeutic methods where the number,activation and/or survival of the engineered immune cells endowed with aCAR is controlled by using an antibody that directly binds to at leastone epitope specific for a monoclonal antibody in the CD123 CARs at thecell surface.

The present invention encompasses an embodiment disclosing an engineeredimmune cell (TCR KO and/or dck KO) expressing individually any one ofthe CD123 specific CAR discloses herein, preferably one of the followingsequences SEQ ID NO 188 to SEQ ID NO 197.

The present invention encompasses an embodiment disclosing vectorsencoding allowing the preparation of engineered immune cell (TCR KOand/or dck KO) expressing individually any (each) one of the CD123specific CAR discloses above. In particular the present inventionencompasses an embodiment disclosing vectors encoding (each) one of theCD123 specific CAR discloses above, preferably comprising a backbone.

The present invention encompasses an embodiment disclosing vectorsencoding allowing the preparation of engineered immune cell (TCR KOand/or dck KO) expressing individually any (each) one of the CD123specific CAR discloses above, preferably one of the following sequencesSEQ ID NO 188 to SEQ ID NO 197.

The present invention encompasses an embodiment disclosing apharmaceutical composition comprising an engineered immune cell (TCR KOand/or dck KO) expressing individually any (each) one of the CD123specific CAR discloses above and a pharmaceutically acceptable vehicle.

The present invention encompasses an embodiment disclosing apharmaceutical composition comprising an engineered immune cell (TCR KOand/or dck KO) expressing individually any (each) one of the CD123specific CAR discloses above and a pharmaceutically acceptable vehiclefor use as a medicament.

The present invention encompasses an embodiment disclosing apharmaceutical composition comprising between from 104 to from 1010/kgengineered immune cells (TCR KO and/or dck KO) expressing individuallyany (each) one of the CD123 specific CAR discloses above and apharmaceutically acceptable vehicle for use as a medicament.

The present invention discloses an engineered immune cell (TCR KO and/ordck KO) expressing a specific CD123 CAR having a polypeptide structureV3 as illustrated in FIG. 2, and described above wherein said CD123 CARhas at least 80% sequence identity with SEQ ID NO. 31.

The present invention discloses an engineered immune cell (TCR KO and/ordck KO) expressing a specific CD123 CAR having a polypeptide structureV3 as illustrated in FIG. 2, and described above wherein said CD123 CARhas at least 80% sequence identity with SEQ ID NO. 32.

The present invention discloses an engineered immune cell (TCR KO and/ordck KO) expressing a specific CD123 CAR having a polypeptide structureV3 as illustrated in FIG. 2, and described above wherein said CD123 CARhas at least 80% sequence identity with SEQ ID NO. 33.

The present invention discloses an engineered immune cell (TCR KO and/ordck KO) expressing a specific CD123 CAR having a polypeptide structureV3 as illustrated in FIG. 2, and described above wherein said CD123 CARhas at least 80% sequence identity with SEQ ID NO. 160.

The present invention discloses an engineered immune cell (TCR KO and/ordck KO) expressing a specific CD123 CAR having one of the followingsequences SEQ ID NO 188 to SEQ ID NO 197.

The present invention discloses an engineered immune cell (TCR KO and/ordck KO) expressing a CD123 CAR as described above, wherein said extracellular ligand binding-domain VH and VL from a monoclonal anti-CD123antibody respectively comprise at least one of the following sequences:

(Variant VH1: SEQ ID NO. 24):EVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (Variant VH2: SEQ ID NO. 25):EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (Variant VH3: SEQ ID NO. 26):EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (Variant VH4: SEQ ID NO. 27):EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (Variant VH5: SEQ ID NO. 28):EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (Variant VH6: SEQ ID NO. 29):EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (Variant VH7: SEQ ID NO. 30):EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSS, Variant VH8EVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS Variant VH9:EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS Variant VH10:EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS Variant VH11:EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSand one of the following sequences:

Variant VL1: SEQ ID NO. 18):MADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,Variant VL2: SEQ ID NO. 19):MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,Variant VL3: SEQ ID NO. 20):MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,Variant VL4: SEQ ID NO. 21):MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,Variant VL5: SEQ ID NO. 22):MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR, andVariant VL6: SEQ ID NO. 23):MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,Variant VL1a: DIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQ GTKVEIKR VariantVL2a: DIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQ GTKVEIKR VariantVL3a: DIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQ GTKVEIKR VariantVL4a: DIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQ GTKVEIKR VariantVL6a: DIQMTQSPSSVSASVGDRVTITCKASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQ GTKVEIKR VariantVL7a: DIQMTQSPSSVSASVGDRVTITCKASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYYSTPWTFGQ GTKVEIKR VariantVL8a: DIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYYSTPWTFGQ GTKVEIKR VariantVL9a: DIQMTQSPSSVSASVGDRVTITCKASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPDRFSGSGSGTDFTLTISSLQPEDLATYYCQQYYSTPWTFGQ GTKVEIKR

The present invention discloses an engineered immune cell (TCR KO and/ordck KO) expressing a CD123 specific CAR as described above, wherein saidextra cellular ligand binding-domain VH and VL from a monoclonalanti-CD123 antibody respectively comprise at least one of the followingsequences:

(SEQ ID NO. 24) EVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 25)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 26)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 27)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 28)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 29)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 30)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 18)MADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR, (SEQID NO. 19) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR, (SEQID NO. 20) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR, (SEQID NO. 21) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR, (SEQID NO. 22) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR, and(SEQ ID NO. 23) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR, or acombination thereof.

Advantageously, the present invention discloses an engineered immunecell (TCR KO and/or dck KO) expressing a CD123 specific CAR as describedabove, wherein said extra cellular ligand binding-domain VH and VL froma monoclonal anti-CD123 antibody respectively comprise at least one ofthe following sequences:

(SEQ ID No 24) EVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS,. (SEQ ID No 25)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS,. (SEQ ID No 26)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS,. (SEQ ID No 27)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS,. (SEQ ID No 28)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS,. (SEQ ID No 29)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS,. (SEQ ID No 30)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSS,.and at least one of the following sequences:

(SEQ ID NO 18) MADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR,. (SEQID NO 172) GCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR huK43-VH9/VL6 (SEQ ID NO 173)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCKASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR huK43-VH10/VL3 (SEQ ID NO 174)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR huK43-VH9/VL8 (SEQ ID NO 175)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR huK43-VH2/VL3 (SEQ ID NO 176)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR huK43-VH10/VL9 (SEQ ID NO 177)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCKASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPDRFSGSGSGTDFTLTISSLQPEDLATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR huK43-VH9/VL3 (SEQ ID NO 178)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR huK43-VH2/VL1 (SEQ ID NO 179)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

In another embodiment the present invention provides an engineeredimmune cell endowed with the following CAR

V5 huK43-VH10/VL1 (SEQ ID NO 180)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPRhuK43-VH9/VL6 (SEQ ID NO 181)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCKASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTSPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPRhuK43-VH10/VL3 (SEQ ID NO 182)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTSPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPRhuK43-VH9/VL8 (SEQ ID NO 183)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPRhuK43-VH2/VL3 (SEQ ID NO 184)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYVGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPRhuK43-VH10/VL9 (SEQ ID NO 185)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLTRSKADGYTTETAASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCKASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPDRFSGSGSGTDFTLTISSLQPEDLATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPRhuK43-VH9/VL3 (SEQ ID NO 186)EVQLVESGGGLVQPGRSLRLSCTASGFTEFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSQSTAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTFITCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPRhuK43-VH2/VL1 (SEQ ID NO 187)EVQLVESGGGLVQPGRSLRLSCTASGETFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR.

The present invention discloses an engineered immune cell (TCR KO and/ordck KO) expressing a CD123 specific CAR as described above, wherein saidstructure V3 (see FIG. 2) comprises a CD8 alpha hinge and a CD8 alphatransmembrane domain, preferably and no CD28 sequence.

The present invention discloses an engineered immune cell (TCR KO and/ordck KO) expressing a CD123 specific CAR as described above, wherein saidstructure V3 comprises a CD8 alpha hinge, a 4-1BB cytoplasmic domain anda CD8 alpha transmembrane domain.

The present invention discloses an engineered immune cell (TCR KO and/ordck KO) expressing a CD123 specific CAR as described above, wherein saidstructure V3 comprises a CD8 alpha hinge and a 4-1BB transmembranedomain and no sequence from CD28.

The present invention discloses an engineered immune cell expressing aCD123 specific CAR as above and further comprising another extracellularligand binding domain which is not specific for CD123. In a preferredembodiment, another extracellular ligand binding domain which is notspecific for CD123 is a suicide domain, more preferably more preferablya suicide domain as any one disclosed in patent application PA 201570044 table 2).

The present invention discloses an engineered immune cell (TCR KO and/ordck KO) expressing a CD123 specific CAR as described above, wherein saidCD123 specific CAR comprises a suicide domain comprising at least one ofSEQ ID NO.: 161, preferably at least two SEQ ID NO.: 161, bound by alinker Li comprising G and S.

In one embodiment said suicide domain is integrated into the hingedomain, of a CD123 CAR of the invention. In one more preferredembodiment, the present invention provides a CD123 CAR of SEQ ID NO.:160or having at least 95% identity with SEQ ID NO:160.

Other suicide domains as those described in table 2 of patentapplication PA201570044 which is incorporated herein by reference in itsentirety are suitable for the present invention.

The present invention discloses an engineered immune cell as above,wherein expression of at least one MHC protein, preferably β2m or HLA,is suppressed in said engineered immune cell. β2m stands for beta 2microglobulin and HLA for human leukocyte antigen. The MHC protein is aMHC protein of Class I or of class II.

The present invention discloses an engineered immune cell as above,wherein said engineered immune cell is engineered to confer resistanceto at least one immune suppressive drug, chemotherapy drug, oranti-cancer drug, preferably to purine analogs.

The present invention discloses a composition comprising apharmaceutically acceptable vehicle and any one of the engineered immunecell (TCR KO and/or dck KO) expressing a CD123 specific CAR as describedabove.

The present invention discloses a composition comprising apharmaceutically acceptable vehicle and any one of the engineered immunecell (TCR KO and/or dck KO) expressing a CD123 specific CAR as describedabove and another drug, preferably a purine analogues and morepreferably a FLAG treatment.

Examples of purine analogues according to the invention may bepentostatin, fludarabine 2-deoxyadenosine, cladribine, clofarabine,Nelarabine, preferably pentostatin, fludarabine monophosphate, and2-chlorodeoxyadenosine (2-CDA).

Examples of FLAG treatments that may be associated with the CD123 Tcells of the invention are as follows: Standard FLAG without additions,FLAG-IDA, Mito-FLAG, FLAMSA.

An Example of FLAG treatment according to the invention may be StandardFLAG without additions

Drug Dose Mode Days (FL)udarabine 30 mg/m² a IV infusion over Days 1-530 min, every 12 hours in 2 day divided doses (A)ra-C 2000 mg/m² IVinfusion over 4 hours, Days 1-5 every 12 hours in 2 divided doses,starting 4 hours after the end of fludarabine infusion (G)-CSF 5 μg/kgSC From day 6 till neutrophil recovery FLAG-IDA (FL)udarabine 30 mg/m² aIV infusion over Days 1-5 30 min, every 12 hours in 2 day divided doses2000 mg/m² a IV infusion over Days 1-5 (A)ra-C 4 hours, every 12 hoursin 2 day divided doses, starting 4 hours after the end of fludarabineinfusion (IDA)rubicin 10 mg/m² IV bolus Days 1-3 (G)-CSF 5 μg/kg SC Fromday 6 till neutrophil recovery Mito-FLAG (FL)udarabine 30 mg/m² IVinfusion over 30 min, Days 1-5 every 12 hours in 2 divided doses (A)ra-C2000 mg/m² IV infusion over 3 hours, Days 1-5 every 12 hours in 2divided doses, starting 4 hours after the end of fludarabine infusion(Mito)xantrone 7 mg/m² IV infusion Days 1, 3 and 5 (G)-CSF 5 μg/kg SCFrom day 6 till neutrophil recovery FLAMSA (FL)udarabine 30 mg/m² IVinfusion over 30 min, Days 1-4 every 12 hours in 2 divided doses (A)ra-C2000 mg/m² IV infusion over 4 hours, Days 1-4 every 12 hours in 2divided doses, starting 4 hours after the end of fiudarabine infusion(AMSA)crine 100 mg/m² IV infusion Days 1-4 Filgrastim 5 μg/kg SC Fromtransplant day (or from day 5 if FLAMSA is not a part of conditioning)till neutrophil recovery

The following combination treatments are disclosed herein:

The present invention discloses an engineered immune cell as any onedescribed above, a composition comprising said engineered immune cell asdisclosed above, for use in therapy.

The present invention discloses an engineered immune cell of theinvention, a composition comprising said engineered immune cell asdisclosed above, for use in therapy as above, wherein the patient is ahuman.

The present invention discloses an engineered immune cell, a compositioncomprising said engineered immune cell as disclosed above, for use intherapy as above, wherein the condition is a pre-malignant or malignantcancer condition characterized by CD123-expressing cells.

The present invention discloses an engineered immune cell, a compositioncomprising said engineered immune cell as disclosed above, for use intherapy as above, wherein the condition is a condition which ischaracterized by an overabundance of CD123-expressing cells.

The present invention discloses an engineered immune cell, a compositioncomprising said engineered immune cell as disclosed above, for use intherapy as above, wherein the malignant cancer condition is ahaematological cancer condition.

The present invention discloses an engineered immune cell, a compositioncomprising said engineered immune cell as disclosed above, for use intherapy as above, wherein the haematological cancer condition isleukemia or malignant lymphoproliferative disorders.

The present invention discloses an engineered immune cell, a compositioncomprising said engineered immune cell as disclosed above, for use intherapy as above, wherein said leukemia is selected from the groupconsisting of acute myelogenous leukemia, chronic myelogenous leukemia,myelodysplastic syndrome, acute lymphoid leukemia, chronic lymphoidleukemia, and myelodysplastic syndrome.

The present invention discloses an engineered immune cell, a compositioncomprising said engineered immune cell as disclosed above, for use intherapy as above, wherein the leukemia is acute myelogenous leukemia(AML), preferably refractory/relapsed AML.

In one embodiment, the present invention discloses an engineered immunecell, a composition comprising said engineered immune cell as disclosedabove, for use in therapy as above, wherein said hematologic cancer is amalignant lymphoproliferative disorder.

The present invention discloses an engineered immune cell for use intherapy as above, wherein said malignant lymphoproliferative disorder islymphoma.

The present invention discloses an engineered immune cell for use intherapy as above, wherein said lymphoma is selected from the groupconsisting of multiple myeloma, non-Hodgkin's lymphoma, Burkitt'slymphoma, and follicular lymphoma (small cell and large cell).

CD123 CAR of SEQ ID No 31 or SEQ ID No 32 or SEQ ID No 160 expressed inTCR KO and dck KO T cells of the invention, from 10⁴ to 10⁸ cells/kg, incombination with a FLAG treatment without addition, for use in thetreatment of AML, preferably refractory relapsed AML.

CD123 CAR of SEQ ID No 31 or SEQ ID No 32 or SEQ ID No 160 expressed inTCR KO and dck KO T cells of the invention, from 10⁴ to 10⁸ cells/kg, incombination with a FLAG treatment without addition, for use in thetreatment of BPDCN.

CD123 CAR of SEQ ID No 31 or SEQ ID No 32 or SEQ ID No 160 expressed inTCR KO and dck KO T cells of the invention from 10⁴ to 10⁸ cells/kg incombination with a FLAG treatment without addition for use as atreatment before bone marrow transplant as a bridge of transplant.

CD123 CAR of SEQ ID No 31 or SEQ ID No 32 or SEQ ID No 160 expressed inTCR KO and dck KO T cells of the invention (from 104 to 108 cells/kg) incombination with fludarabine (from 20 mg/kg to 50 mg/kg), for use in thetreatment of AML, preferably refractory relapsed AML.

CD123 CAR of SEQ ID No 31 or SEQ ID No 32 or SEQ ID No 160 expressed inTCR KO and dck KO T cells of the invention (from 10⁴ to 10⁸ cells/kg) incombination with fludarabine (from 20 mg/kg to 50 mg/kg), for use in thetreatment of BPDCN.

CD123 CAR of SEQ ID No 31 or SEQ ID No 32 or SEQ ID No 160 expressed inTCR KO and dck KO T cells of the invention (from 104 to 108 cells/kg) incombination with fludarabine (from 20 mg/kg to 50 mg/kg), for use as atreatment before bone marrow transplant as a bridge of transplant.

The present invention discloses a method of impairing a hematologiccancer cell comprising contacting said hematologic cancer cell with anengineered cell according to the invention in an amount effective tocause impairment of said cancer cell (from 10⁴ to 10⁸ cells/kg).

The present invention discloses a method of engineering an immune cellcomprising:

-   -   1. Providing an immune cell from a donor,    -   2. Knocking out the TCR gene,    -   3. Expressing at the surface of said cell at the CD123 specific        chimeric antigen receptor according to the invention as any one        of the above.

A donor may the patient suffering a cancer himself (for autologousadoptive transfer) or another individual (for adoptive transfer ofallogenic T cells). The present invention discloses a method ofengineering an immune cell as above comprising:

-   -   1. Providing an immune cell from a donor,    -   2. Knocking out the TCR gene, and the dck gene    -   3. Expressing at the surface of said cell the CD123 specific        chimeric antigen receptor according to any one of the above by        introducing into said cell at least one polynucleotide encoding        said CD123 specific chimeric antigen receptor,

In a more preferred embodiment, said method comprises expressing at thecell surface a suicide domain.

The present invention discloses a method of engineering an immune cellas above comprising:

-   -   1. Providing an immune cell,    -   2. Knocking out a TCR gene, using half TALE-nuclease TALEN of        SEQ ID NO: 16 and SEQ ID NO: 17 and the dck gene    -   3. Expressing at the surface of said cell any one of CD123        specific chimeric antigen receptor according to the above by        introducing into said cell at least one polynucleotide encoding        said CD123 specific chimeric antigen receptor.

In a preferred embodiment, said method comprises expressing at the cellsurface a suicide domain, preferably a suicide domain recognized by oneof the following antibodies ibritumomab, tiuxetan, muromonab-CD3,tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab,infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol,daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab,palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab,adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab,ofatumumab, panitumumab, QBEND-10 and ustekinumab.

In another embodiment said method further comprises a step of bindingsaid engineered immune cell of the invention to a specific monoclonalantigen as those disclosed herein selected from ibritumomab, tiuxetan,muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin,cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumabpegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab,omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab,vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab,ipilimumab, ofatumumab, panitumumab, QBEND-10 and ustekinumab.

The present invention discloses a method of treating a subject in needthereof comprising:

-   -   1. Providing an immune cell expressing at the surface a CD123        specific Chimeric Antigen Receptor according to any one of the        above or a composition comprising it    -   2. Administrating said immune cells to said patient. In a        preferred embodiment, said composition further comprises a        purine analogue, fludarabine

In another embodiment, said composition is associated to a FLAGtreatment, a FLAG treatment without addition.

In one embodiment said subject in need thereof suffers AML, preferablyrefractory relapsed AML, BPDNL, or must have bone marrowtransplantation.

The present invention discloses a method of treating a subject in needthereof as above, wherein an immune cell is provided from a donor.

The present invention discloses a method of treating a subject in needthereof as above, wherein said immune cell is provided from the patienthimself.

CD123 Specific Chimeric Antigen Receptors

The present invention relates to new anti-CD123 chimeric antigenreceptor (CAR) comprising an extracellular ligand-binding domain from orderived from KLON 43 antibody, a transmembrane domain from CD8 alpha, ahinge from CD8 alpha or from FcγRIIIα, a suicide domain and a signalingtransducing domain. In a preferred embodiment said suicide domain isintegrated into the hinge domain. In a more preferred embodiment saidsuicide domain comprises at least two sequences of SEQ ID NO 161integrated into the hinge domain.

In a preferred embodiment, said anti-CD123 CAR of the invention is apolypeptide of SEQ ID NO.: 31, 32, or 160,

In another embodiment, said anti-CD123 CAR of the invention is apolypeptide of SEQ ID NO.: 34 to SEQ ID NO.:159, SEQ ID NO.:34 to SEQ IDNO.:117, SEQ ID NO.:76 to SEQ ID NO.:117.

The term “extracellular ligand-binding domain” as used herein is definedas an oligo- or polypeptide that is capable of binding CD123.Preferably, the extracellular ligand-binding domain may be chosen torecognize CD123 that acts as a cell surface marker on target cellsassociated with a particular disease state. More preferably, theextracellular ligand-binding domain may be chosen to recognize CD123that acts as a cell surface marker on target cells associated with AML,BPDCN or a CD123-expressing cell involved in a cancer state.

In a preferred embodiment, said extracellular ligand-binding domaincomprises a single chain antibody fragment (scFv) comprising the light(VL) and the heavy (VH) variable fragment of a target antigen specificmonoclonal anti CD-123 antibody KLON 43 joined by a flexible linker.Said V_(L) and VH are preferably selected from the sequences disclosedin Table 1 to 2, more preferably an scfv comprising a VH, a linker andVL from or derived from Klon43 (humanized VH and VL as described intable 2). They are preferably linked together by a flexible linker ofsequence (GGGGS)n wherein n=1 to 4, more preferably n=3 comprising thesequence SEQ ID NO. 10. In other words, said CARs preferentiallycomprise an extracellular ligand-biding domain comprising a polypeptidesequence 100% identity with an amino acid sequence selected from thegroup consisting of SEQ ID NO: 12 for VH and SEQ ID NO: 11 for VL andSEQ ID NO: 18 to SEQ ID NO: 30 for humanized fragments (see Table 2).

By the term “recombinant antibody” as used herein, is meant an antibodyor antibody fragment which is generated using recombinant DNAtechnology, such as, for example, an antibody or antibody fragmentexpressed by a bacteriophage, a yeast expression system or a mammaliancell expression system. The term should also be construed to mean anantibody or antibody fragment which has been generated by the synthesisof a DNA molecule encoding the antibody or antibody fragment and whichDNA molecule expresses an antibody or antibody fragment protein, or anamino acid sequence specifying the antibody or antibody fragment,wherein the DNA or amino acid sequence has been obtained usingrecombinant or synthetic DNA or amino acid sequence technology which isavailable and well known in the art.

As used herein, the term “conservative sequence modifications” or“humanization” or “humanized antibody” or “humanized antibody fragment”,“humanized VH or humanized VL” is intended to refer to amino acidmodifications that do not significantly affect or alter the bindingcharacteristics of the CAR and/or that do not significantly affect theactivity of the CAR containing the modified amino acid sequence andreduce or abolish a human anti-mouse antibody (HAMA) response.

In a preferred embodiment, amino acid modifications significantlyimprove the binding characteristics of the CAR and/or significantlyimprove the activity of the CAR containing the modified amino acidsequence and reduce or abolish a human anti-mouse antibody (HAMA)response.

Such conservative modifications include amino acid substitutions,additions and deletions in said antibody fragment in said CAR and/or anyof the other parts of said CAR molecule. Modifications can be introducedinto an antibody, into an antibody fragment or in any of the other partsof the CAR molecule of the invention by standard techniques known in theart, such as site-directed mutagenesis, PCR-mediated mutagenesis or byemploying optimized germline sequences.

Conservative amino acid substitutions are ones in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one ormore amino acid residues within a CAR of the invention can be replacedwith other amino acid residues from the same side chain family and thealtered CAR can be tested for the ability to bind CD 123 using thefunctional assays described herein.

In one embodiment said scfv comprises at least one, preferably twoepitopes binding to a monoclonal antibody. Examples of such epitopes aredisclosed in table 7.

The signal transducing domain or intracellular signaling domain of a CARaccording to the present invention is responsible for intracellularsignaling following the binding of extracellular ligand binding domainto the target resulting in the activation of the immune cell and immuneresponse. In other words, the signal transducing domain is responsiblefor the activation of at least one of the normal effector functions ofthe immune cell in which the CAR is expressed. For example, the effectorfunction of a T cell can be a cytolytic activity or helper activityincluding the secretion of cytokines. Thus, the term “signal transducingdomain” refers to the portion of a protein which transduces the effectorsignal function signal and directs the cell to perform a specializedfunction.

Preferred examples of signal transducing domain for use in a CAR can bethe cytoplasmic sequences of the T cell receptor and co-receptors thatact in concert to initiate signal transduction following antigenreceptor engagement, as well as any derivate or variant of thesesequences and any synthetic sequence that has the same functionalcapability. Signal transduction domain comprises two distinct classes ofcytoplasmic signaling sequence, those that initiate antigen-dependentprimary activation, and those that act in an antigen-independent mannerto provide a secondary or co-stimulatory signal. Primary cytoplasmicsignaling sequence can comprise signaling motifs which are known asimmunoreceptor tyrosine-based activation motifs of ITAMs. ITAMs are welldefined signaling motifs found in the intracytoplasmic tail of a varietyof receptors that serve as binding sites for syk/zap70 class tyrosinekinases. Examples of ITAM used in the invention can include as nonlimiting examples those derived from TCRzeta, FcRgamma, FcRbeta,FcRepsilon, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b andCD66d. In a preferred embodiment, the signaling transducing domain ofthe CAR can comprise the CD3zeta signaling domain which has amino acidsequence with at least 70%, preferably at least 80%, more preferably atleast 90%, 95% 97% or 99% or 100% sequence identity with amino acidsequence selected from the group consisting of SEQ ID NO: 9.

In particular embodiment the signal transduction domain of the CAR ofthe present invention comprises a co-stimulatory signal molecule. Aco-stimulatory molecule is a cell surface molecule other than an antigenreceptor or their ligands that is required for an efficient immuneresponse. “Co-stimulatory ligand” refers to a molecule on an antigenpresenting cell that specifically binds a cognate co-stimulatorymolecule on a T-cell, thereby providing a signal which, in addition tothe primary signal provided by, for instance, binding of a TCR/CD3complex with an MHC molecule loaded with peptide, mediates a T cellresponse, including, but not limited to, proliferation activation,differentiation and the like. A co-stimulatory ligand can include but isnot limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL,OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, an agonist or antibodythat binds Toll ligand receptor and a ligand that specifically bindswith B7-H3. A co-stimulatory ligand also encompasses, inter alia, anantibody that specifically binds with a co-stimulatory molecule presenton a T cell, such as but not limited to, CD27, CD28, 4-1BB, OX40, CD30,CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2,CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83. A“co-stimulatory molecule” refers to the cognate binding partner on aT-cell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the cell, such as, but notlimited to proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and Toll ligand receptor.Examples of costimulatory molecules include CD27, CD28, CD8, 4-1BB(CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associatedantigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand thatspecifically binds with CD83.

In one embodiment, the signal transduction domain of the CAR of thepresent invention in particular the co-stimulatory molecules do notinclude and CD28 (NP_006130.1).

In one preferred embodiment, the CAR of the present invention does notinclude a sequence of human CD28 (NP_006130.1) and/or from any otherCD28.

In another preferred embodiment, the signal transduction domain of theCAR of the present invention comprises a part of co-stimulatory signalmolecule selected from the group consisting of fragments of 4-1BB(GenBank: AAA53133.). In particular the signal transduction domain ofthe CAR of the present invention comprises amino acid sequence whichcomprises at least 70%, preferably at least 80%, more preferably atleast 90%, 95% 97% or 99% sequence identity with amino acid sequenceselected from the group consisting of SEQ ID NO: 8.

A CAR according to the present invention is expressed on the surfacemembrane of the cell. Thus, such CAR further comprises a transmembranedomain. The distinguishing features of appropriate transmembrane domainscomprise the ability to be expressed at the surface of a cell,preferably in the present invention an immune cell, in particularlymphocyte cells or Natural killer (NK) cells, and to interact togetherfor directing cellular response of immune cell against a predefinedtarget cell. The transmembrane domain can be derived either from anatural or from a synthetic source. The transmembrane domain can bederived from any membrane-bound or transmembrane protein. Asnon-limiting examples, the transmembrane polypeptide can be a subunit ofthe T-cell receptor such as α, β, γ or δ, polypeptide constituting CD3complex, IL2 receptor p55 (α chain), p75 (β chain) or γ chain, subunitchain of Fc receptors, in particular Fcγ receptor III or CD proteins.Alternatively the transmembrane domain can be synthetic and can comprisepredominantly hydrophobic residues such as leucine and valine.

In a preferred embodiment said transmembrane domain (TM) is derived fromthe human CD8 alpha chain (e.g. NP_001139345.1), IgG1, IgG4, FcγRIIIα.

In a more preferred embodiment said TM domain comprises a sequence orpart of the SEQ ID NO 6. A CD123 CAR according to the inventiongenerally further comprises a transmembrane domain (TM) from CD8α,showing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identity with the polypeptides of SEQ ID NO. 6.

In one embodiment the CD123 CAR of the invention does not comprise a TMdomain from 4-1BB, preferably of sequence IISFFLALTSTALLFLLFFLTLRFSVV(SEQ ID NO. 7) In a more preferred embodiment said Hinge is of SEQ ID NO171.

A CAR according to the present invention comprises a hinge regionbetween said extracellular ligand-binding domain and said transmembranedomain. The term “hinge region” used herein generally means any oligo-or polypeptide that functions to link the transmembrane domain to theextracellular ligand-binding domain. In particular, hinge region areused to provide more flexibility and accessibility for the extracellularligand-binding domain. A hinge region may comprise up to 300 aminoacids, preferably 10 to 100 amino acids and most preferably 25 to 50amino acids. Hinge region may be derived from all or part of naturallyoccurring molecules, such as from all or part of the extracellularregion of CD8, CD4, or from all or part of an antibody constant region.

In one embodiment, said hinge region comprises at least one epitopesrecognized by a monoclonal antibody, as disclosed in table 7.

In one embodiment, said hinge region comprises at least two sequences ofSEQ ID No 161 and preferably is of SEQ ID No 171.

In one embodiment said CD123 CAR of the invention comprises at least onesequence of SEQ ID No 161 in the scfv.

In one embodiment said CD123 CAR of the invention comprises twosequences of SEQ ID No 161 and a sequence of SEQ ID NO 169 in the scfv,preferably at the N-terminal end of the scfv.

Alternatively, the hinge region may be a synthetic sequence thatcorresponds to a naturally occurring hinge sequence, or may be anentirely synthetic hinge sequence. In a preferred embodiment said hingedomain comprises a part of human CD8 alpha chain, FcγRIIIα receptor orIgG1 respectively referred to in this specification as SEQ ID NO. 3, SEQID NO. 4 and SEQ ID NO. 5, or hinge polypeptides which displaypreferably at least 80%, more preferably at least 90%, 95% 97% or 99%sequence identity with these polypeptides.

In a more preferred embodiment, said hinge domain comprises a part ofhuman CD8 alpha chain, or of FcγRIIIα receptor, more preferably saidhinge domain comprises a sequence of SEQ ID NO. 3 or of SEQ ID NO. 4 orwith at least 80%, more preferably at least 90%, 95% 97% or 99% sequenceidentity with SEQ ID NO. 3 or SEQ ID NO. 4.

Other Additional Scfv

Downregulation or mutation of target antigens is commonly observed incancer cells, creating antigen-loss escape variants. Thus, to offsettumor escape and render immune cell more specific to target, the CD123specific CAR according to the invention can comprise anotherextracellular ligand-binding domains, to simultaneously bind differentelements in target thereby augmenting immune cell activation andfunction. In one embodiment, the extracellular ligand-binding domainscan be placed in tandem on the same transmembrane polypeptide, andoptionally can be separated by a linker. In another embodiment, saiddifferent extracellular ligand-binding domains can be placed ondifferent transmembrane polypeptides composing the CAR. In anotherembodiment, the present invention relates to a population of CARscomprising each one different extracellular ligand binding domains. In aparticular, the present invention relates to a method of engineeringimmune cells comprising providing an immune cell and expressing at thesurface of said cell a population of CAR each one comprising differentextracellular ligand binding domains. In another particular embodiment,the present invention relates to a method of engineering an immune cellcomprising providing an immune cell and introducing into said cellpolynucleotides encoding polypeptides composing a population of CAR eachone comprising different extracellular ligand binding domains. Bypopulation of CARs, it is meant at least two, three, four, five, six ormore CARs each one comprising different extracellular ligand bindingdomains. The different extracellular ligand binding domains according tothe present invention can preferably simultaneously bind differentelements in target thereby augmenting immune cell activation andfunction.

The present invention also relates to an isolated immune cell whichcomprises a population of CARs each one comprising differentextracellular ligand binding domains.

In a preferred embodiment, a CD123 CAR according to the inventioncomprises a polypeptide of SEQ ID NO. 31 or a polypeptide of SEQ ID NO.32, more preferably a CD123 CAR according to the invention comprises apolypeptide with at least 80% identity, preferably 80% to 99% identitywith SEQ ID NO. 31 or a polypeptide having 80 to 99% identity with SEQID NO. 32. Even more preferably a CAR according to the inventioncomprises a polypeptide having 85 to 99% identity with a polypeptide ofSEQ ID NO. 31 or with SEQ ID NO. 32.

In a preferred embodiment, a CD123 CAR according to the inventioncomprises a polypeptide having the following sequences SEQ ID NO. 31.

In a preferred embodiment, a CD123 CAR according to the inventioncomprises a polypeptide having the following sequence selected from SEQID NO. 32, SEQ ID NO. 31 and SEQ ID NO. 160.

In one preferred embodiment, a CAR according to the invention comprisesat least one polypeptide selected from the following sequences:

(SEQ ID NO. 24) EVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 25)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 26)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 27)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 28)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS, (SEQ ID NO. 29)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSS and (SEQ ID NO. 30)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSSand at least one sequence selected from the following sequences

(SEQ ID NO. 18) MADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR, (SEQID NO. 19) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR, (SEQID NO. 20) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR, (SEQID NO. 21) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR, (SEQID NO. 22) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR and(SEQ ID NO. 23) MADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTP WTFGQGTKVEIKR.

In one embodiment, a CD123 CAR according to the invention comprises onepolypeptide selected from the following sequences: SEQ ID NO. 24, SEQ IDNO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, andSEQ ID NO. 30 and a peptide selected from the following sequences: SEQID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22,and SEQ ID NO. 23.

In one embodiment, a CD123 CAR according to the invention comprises apolypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with apolypeptide of SEQ ID NO. 11, or having at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity with a polypeptide of SEQ ID NO. 12.

In a more preferred embodiment, a CD123 CAR according to the inventioncomprises a polypeptide comprising 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity with SEQ ID NO. 11+SEQ ID NO. 10+SEQ ID NO. 12.

According to the invention, the immune cells expressing the anti-CD123CAR of the invention trigger an anti-cancer immune response, no orreduce GVHD and proliferate even in the presence of purine analogue ofFLAG treatment.

In a preferred embodiment, the immune cells expressing the CAR of theinvention endowed with the anti-CD123 CAR of the invention does triggeran immune response which does not comprise a human anti-mouse antibody(HAMA) response.

According to the invention, an efficient amount of the engineered immunecell of the invention can be administered to a patient in need thereofat least once, alone or in combination with another treatment.

Polynucleotides, Vectors

The present invention also relates to polynucleotides, vectors encodingthe above described CAR according to the invention.

The polynucleotide may consist in an expression cassette or expressionvector (e.g. a plasmid for introduction into a bacterial host cell, or aviral vector such as a baculovirus vector for transfection of an insecthost cell, or a plasmid or viral vector such as a lentivirus fortransfection of a mammalian host cell).

In a particular embodiment, the different nucleic acid sequences can beincluded in one polynucleotide or vector which comprises a nucleic acidsequence encoding ribosomal skip sequence such as a sequence encoding a2A peptide. 2A peptides, which were identified in the Aphthovirussubgroup of picornaviruses, causes a ribosomal “skip” from one codon tothe next without the formation of a peptide bond between the two aminoacids encoded by the codons (see (Donnelly and Elliott 2001; Atkins,Wills et al. 2007; Doronina, Wu et al. 2008)). By “codon” is meant threenucleotides on an mRNA (or on the sense strand of a DNA molecule) thatare translated by a ribosome into one amino acid residue. Thus, twopolypeptides can be synthesized from a single, contiguous open readingframe within an mRNA when the polypeptides are separated by a 2Aoligopeptide sequence that is in frame. Such ribosomal skip mechanismsare well known in the art and are known to be used by several vectorsfor the expression of several proteins encoded by a single messengerRNA.

To direct transmembrane polypeptide into the secretory pathway of a hostcell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in polynucleotide sequenceor vector sequence. The secretory signal sequence is operably linked tothe transmembrane nucleic acid sequence, i.e., the two sequences arejoined in the correct reading frame and positioned to direct the newlysynthesized polypeptide into the secretory pathway of the host cell.Secretory signal sequences are commonly positioned 5′ to the nucleicacid sequence encoding the polypeptide of interest, although certainsecretory signal sequences may be positioned elsewhere in the nucleicacid sequence of interest (see, e.g., Welch et al., U.S. Pat. No.5,037,743; Holland et al., U.S. Pat. No. 5,143,830). In a preferredembodiment the signal peptide comprises the amino acid sequence SEQ IDNO: 1 and 2 or at least 90%, 95% 97% or 99% sequence identity with SEQID NO: 1 or 2, preferably SEQ ID NO: 1.

Those skilled in the art will recognize that, in view of the degeneracyof the genetic code, considerable sequence variation is possible amongthese polynucleotide molecules. Preferably, the nucleic acid sequencesof the present invention are codon-optimized for expression in mammaliancells, preferably for expression in human cells. Codon-optimizationrefers to the exchange in a sequence of interest of codons that aregenerally rare in highly expressed genes of a given species by codonsthat are generally frequent in highly expressed genes of such species,such codons encoding the amino acids as the codons that are beingexchanged.

In one embodiment, the different nucleic acid sequences encoding a CD123CAR of the invention can be included in one polynucleotide or vector.

In a more preferred embodiment the claimed invention is directed to avector allowing a stable expression of the CAR of the invention. Stablemeans here that the CAR of the invention is detected at the cell surfaceof engineered cells at least 1 year after injection.

In another embodiment the claimed invention is directed to a vector,allowing a stable expression of a CD123 CAR of the invention, preferablyof SEQ ID NO. 31, SEQ ID NO 32 or SEQ ID NO 160.

In a preferred embodiment, the present invention provides a pCLS27333vector comprising a sequence encoding any one of the CD123 CAR of theinvention preferably a CD123 CAR of SEQ ID NO 31, SEQ ID NO 32 or SEQ IDNO. 160.

Cells

Cell according to the present invention refers to a cell ofhematopoietic origin functionally involved in the initiation and/orexecution of innate and/or adaptative immune response. Cell according tothe present invention is preferably a T-cell obtained from a donor. SaidT cell according to the present invention can be derived from a stemcell. The stem cells can be adult stem cells, embryonic stem cells, moreparticularly non-human stem cells, cord blood stem cells, progenitorcells, bone marrow stem cells, totipotent stem cells or hematopoieticstem cells. In a preferred embodiment, cells are human cells, inparticular human stem cells. In a more preferred embodiment, cells arehuman T cells, in particular human engineered T cells.

Representative human stem cells are CD34+ cells. Said isolated cell canalso be a dendritic cell, killer dendritic cell, a mast cell, a NK-cell,a B-cell or a T-cell selected from the group consisting of inflammatoryT-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes orhelper T-lymphocytes. In another embodiment, said cell can be derivedfrom the group consisting of CD4+T-lymphocytes and CD8+T-lymphocytes. Ina preferred embodiment, said cell can be derived from the groupconsisting of engineered CD4+T-lymphocytes and engineeredCD8+T-lymphocytes.

Prior to expansion and genetic modification of the cells of theinvention, a source of cells can be obtained from a subject through avariety of non-limiting methods. Cells can be obtained from a number ofnon-limiting sources, including peripheral blood mononuclear cells, bonemarrow, lymph node tissue, cord blood, thymus tissue, tissue from a siteof infection, ascites, pleural effusion, spleen tissue, and tumors. Incertain embodiments of the present invention, any number of T-cell linesavailable and known to those skilled in the art, may be used. In anotherembodiment, said cell is preferably derived from a healthy donor. Inanother embodiment, said cell is part of a mixed population of cellswhich present different phenotypic characteristics.

Preferably, isolation and preparation of stem cells does not require thedestruction of at least one human embryo. The immune cells can originatefrom the patient, in view of operating autologous treatments, or fromone or several donors in view of producing allogeneic cells, which canbe used in allogeneic treatments.

More preferably the engineered immune cell of the invention express ananti-CD123 CAR corresponding to SEQ ID NO 31, SEQ ID NO 32, or SEQ ID NO33 at the cell surface, even more preferably the engineered immune cellof the invention express an humanized anti-CD123 CAR corresponding tohumanized SEQ ID NO 31, SEQ ID NO 32, or SEQ ID NO 32.

In one embodiment the engineered immune cell of the invention express ananti-CD123 CAR corresponding to SEQ ID NO. 34 to SEQ ID NO. 159,preferably SEQ ID NO. 34 to SEQ ID NO. 117, more preferably SEQ ID NO.76 to SEQ ID NO. 117.

Among these anti-CD123 CARs, those comprising

VH1/VL1, VH1/VL3; VH1/VL4; VH2/VL2, VH2/VL3, VH2/VL4; VH3/VL1, VH3/VL2,VH3/VL3, VH3/VL4 VH4/VL1, VH4/VL2, VH4/VL3, VH4/VL4

are preferred;

VH1/VL1, VH1/VL3; VH2/VL4; VH3/VL1, VH3/VL2, VH3/VL3,

VH4/VL1, VH4/VL2, VH4/VL3, VH4/VL4 are more preferred,and those comprising VH3/VL2, VH4/VL1, VH4/VL2, VH4/VL3, VH4/VL4 areeven more preferred.

The most preferred are those having a humanized Klone 43 (huK43) and

VH10a/VL1a VH9a/VL6a VH10a/VL3a VH9a/VL8a VH2a/VL3a VH10a/VL9a VH9a/VL3aVH2a/VL1a

In one embodiment the engineered immune cell of the invention express ananti-CD123 CAR corresponding to SEQ ID NO. 160 and SEQ ID NO. 172 to SEQID NO. 187

Methods of Engineering Immune Cells Endowed with CARs:

The present invention encompasses the method of preparing immune cellsfor immunotherapy comprising introducing ex-vivo into said immune cellsthe polynucleotides or vectors encoding the CD123 CAR previouslydescribed in WO2014/130635 WO2013176916, WO2013176915 and incorporatedherein by reference.

In a preferred embodiment, said polynucleotides are included inlentiviral vectors in view of being stably expressed in the immunecells.

In a more preferred embodiment, said polynucleotide is included in alentiviral vector in view of being stably expressed in the immune cells.

According to further embodiments, said method further comprises the stepof genetically modifying said cell to make them more suitable forallogeneic transplantation and to reduce GVHD response.

Modifying T-Cell by Inactivating at Least One Gene Encoding a T-CellReceptor (TCR) Component.

According to a first aspect, the immune cell can be made lessallogeneic, for instance, by inactivating at least one gene expressingone or more component of T-cell receptor (TCR) as described in WO2013/176915, which can be combined with the inactivation of a geneencoding or regulating HLA or β2m protein expression. Accordingly therisk of graft versus host syndrome and graft rejection is significantlyreduced.

Accordingly, when the immune cells are T-cells, the present inventionalso provides methods to engineer T-cells that are less allogeneic.

Methods of making cells less allogenic can comprise the step ofinactivating at least one gene encoding a T-Cell Receptor (TCR)component, in particular TCRalpha, TCRbeta genes.

Methods disclosed in WO2013/176915 to prepare CAR expressing immune cellsuitable for allogeneic transplantation, by inactivating one or morecomponent of T-cell receptor (TCR), are all incorporated herein byreference.

The present invention encompasses an anti-CD123 CAR expressing immunecell wherein at least one gene expressing one or more component ofT-cell receptor (TCR) has been inactivated. Thus, the present inventionprovides an anti-CD123 CAR expressing T cell wherein the CAR is derivedfrom Klon 43, in particular having at least 80% identity with SEQ ID No31 and wherein at least one gene expressing one or more component ofT-cell receptor (TCR) is inactivated.

The present invention encompasses an anti-CD123 CAR expressing immunecell wherein at least one gene expressing one or more component ofT-cell receptor (TCR) has been inactivated. Thus, the present inventionprovides an anti-CD123 CAR expressing T cell wherein the CAR is derivedfrom Klon 43, in particular having at least 80% identity with SEQ ID No32 and wherein at least one gene expressing one or more component ofT-cell receptor (TCR) is inactivated.

The present invention encompasses an anti-CD123 CAR expressing immunecell wherein at least one gene expressing one or more component ofT-cell receptor (TCR) has been inactivated. Thus, the present inventionprovides an anti-CD123 CAR expressing T cell wherein the CAR is derivedfrom Klon 43, in particular having at least 80% identity with SEQ ID No160 and wherein at least one gene expressing one or more component ofT-cell receptor (TCR) is inactivated.

According to the invention, anti-CD123 CAR immune cells with one or morecomponent of T-cell receptor (TCR) inactivated, are intended to be usedas a medicament.

By inactivating a TCR gene it is intended that the gene of interest isnot expressed in a functional protein form. In particular embodiments,the genetic modification of the method relies on the expression, inprovided cells to engineer, of one rare-cutting endonuclease such thatsaid rare-cutting endonuclease specifically catalyzes cleavage in onetargeted gene thereby inactivating said targeted gene. The nucleic acidstrand breaks caused by the rare-cutting endonuclease are commonlyrepaired through the distinct mechanisms of homologous recombination ornon-homologous end joining (NHEJ). However, NHEJ is an imperfect repairprocess that often results in changes to the DNA sequence at the site ofthe cleavage. Mechanisms involve rejoining of what remains of the twoDNA ends through direct re-ligation (Critchlow and Jackson 1998) or viathe so-called microhomology-mediated end joining (Betts, Brenchley etal. 2003; Ma, Kim et al. 2003). Repair via non-homologous end joining(NHEJ) often results in small insertions or deletions and can be usedfor the creation of specific gene knockouts. Said modification may be asubstitution, deletion, or addition of at least one nucleotide. Cells inwhich a cleavage-induced mutagenesis event, i.e. a mutagenesis eventconsecutive to an NHEJ event, has occurred can be identified and/orselected by well-known method in the art. In a particular embodiment,the step of inactivating at least a gene encoding a component of theT-cell receptor (TCR) into the cells of each individual sample comprisesintroducing into the cell a rare-cutting endonuclease able to disrupt atleast one gene encoding a component of the T-cell receptor (TCR). In amore particular embodiment, said cells of each individual sample aretransformed with nucleic acid encoding a rare-cutting endonucleasecapable of disrupting at least one gene encoding a component of theT-cell receptor (TCR), and said rare-cutting endonuclease is expressedinto said cells.

Said rare-cutting endonuclease can be a meganuclease, a Zinc fingernuclease, CRISPR/Cas9 nuclease, Argonaute nuclease, a TALE-nuclease or aMBBBD-nuclease. In a preferred embodiment, said rare-cuttingendonuclease is a TALE-nuclease. By TALE-nuclease is intended a fusionprotein consisting of a DNA-binding domain derived from a TranscriptionActivator Like Effector (TALE) and one nuclease catalytic domain tocleave a nucleic acid target sequence (Boch, Scholze et al. 2009; Moscouand Bogdanove 2009; Christian, Cermak et al. 2010; Cermak, Doyle et al.2011; Geissler, Scholze et al. 2011; Huang, Xiao et al. 2011; Li, Huanget al. 2011; Mahfouz, Li et al. 2011; Miller, Tan et al. 2011;Morbitzer, Romer et al. 2011; Mussolino, Morbitzer et al. 2011; Sander,Cade et al. 2011; Tesson, Usal et al. 2011; Weber, Gruetzner et al.2011; Zhang, Cong et al. 2011; Deng, Yan et al. 2012; Li, Piatek et al.2012; Mahfouz, Li et al. 2012; Mak, Bradley et al. 2012). In the presentinvention new TALE-nucleases have been designed for precisely targetingrelevant genes for adoptive immunotherapy strategies.

Preferred TALE-nucleases recognizing and cleaving the target sequenceare described in PCT/EP2014/075317. In particular, additional catalyticdomain can be further introduced into the cell with said rare-cuttingendonuclease to increase mutagenesis in order to enhance their capacityto inactivate targeted genes. More particularly, said additionalcatalytic domain is a DNA end processing enzyme. Non limiting examplesof DNA end-processing enzymes include 5-3′ exonucleases, 3-5′exonucleases, 5-3′ alkaline exonucleases, 5′ flap endonucleases,helicases, phosphatase, hydrolases and template-independent DNApolymerases. Non limiting examples of such catalytic domain comprise ofa protein domain or catalytically active derivate of the protein domainselected from the group consisting of hExol (EXO1_HUMAN), Yeast Exol(EXO1_YEAST), E. coli Exol, Human TREX2, Mouse TREX1, Human TREX1,Bovine TREX1, Rat TREX1, TdT (terminal deoxynucleotidyl transferase)Human DNA2, Yeast DNA2 (DNA2_YEAST). In a preferred embodiment, saidadditional catalytic domain has a 3′-5′-exonuclease activity, and in amore preferred embodiment, said additional catalytic domain is TREX,more preferably TREX2 catalytic domain (WO2012/058458). In anotherpreferred embodiment, said catalytic domain is encoded by a single chainTREX2 polypeptide. Said additional catalytic domain may be fused to anuclease fusion protein or chimeric protein according to the inventionoptionally by a peptide linker.

Endonucleolytic breaks are known to stimulate the rate of homologousrecombination. Thus, in another embodiment, the genetic modificationstep of the method further comprises a step of introduction into cellsof an exogeneous nucleic acid comprising at least a sequence homologousto a portion of the target nucleic acid sequence, such that homologousrecombination occurs between the target nucleic acid sequence and theexogeneous nucleic acid. In particular embodiments, said exogenousnucleic acid comprises first and second portions which are homologous toregion 5′ and 3′ of the target nucleic acid sequence, respectively. Saidexogenous nucleic acid in these embodiments also comprises a thirdportion positioned between the first and the second portion whichcomprises no homology with the regions 5′ and 3′ of the target nucleicacid sequence. Following cleavage of the target nucleic acid sequence, ahomologous recombination event is stimulated between the target nucleicacid sequence and the exogenous nucleic acid. Preferably, homologoussequences of at least 50 bp, preferably more than 100 bp and morepreferably more than 200 bp are used within said donor matrix. In aparticular embodiment, the homologous sequence can be from 200 bp to6000 bp, more preferably from 1000 bp to 2000 bp. Indeed, shared nucleicacid homologies are located in regions flanking upstream and downstreamthe site of the break and the nucleic acid sequence to be introducedshould be located between the two arms.

Drug Resistant T-Cells

The inventor sought to engineer TCR KO T-cell for immunotherapy, inparticular to engineer TCR KO anti-CD123 CAR of SEQ ID NO 31, of SEQ IDNO 32, or of SEQ ID NO 160 expressing immune cell that can be used incombination with a therapeutic agent (anti-cancer drug).

As used herein, a cell which is “resistant or tolerant” to an agentmeans a cell which has been genetically modified so that the cellproliferates and is active in the presence of an amount of an agent thatinhibits or prevents proliferation of a cell without the geneticmodification.

By inactivating a gene it is intended that the gene of interest is notexpressed in a functional protein form. In particular embodiment, thegenetic modification of the method relies on the expression, in providedcells to engineer, of one rare-cutting endonuclease such that saidrare-cutting endonuclease specifically catalyzes cleavage in onetargeted gene thereby inactivating said targeted gene. In a particularembodiment, the step of inactivating at least one drug sensitizing genecomprises introducing into the cell a rare-cutting endonuclease able todisrupt at least one drug sensitizing gene. In a more particularembodiment, said cells are transformed with nucleic acid encoding arare-cutting endonuclease capable of disrupting a drug sensitizing gene,and said rare-cutting endonuclease is expressed into said cells. Saidrare-cutting endonuclease can be a meganuclease, a Zinc finger nuclease,CRISPR/Cas9 nuclease, A MBBBD-nuclease or a TALE-nuclease. In apreferred embodiment, said rare-cutting endonuclease is a TALE-nuclease.

In a preferred embodiment, a drug sensitizing gene which can beinactivated to confer drug resistance to the T-cell is the humandeoxycytidine kinase (dCK) gene. This enzyme is required for thephosphorylation of the deoxyribonucleosides deoxycytidine (dC),deoxyguanosine (dG) and deoxyadenosine (dA). Purine nucleotide analogs(PNAs) are metabolized by dCK into mono-, di- and tri-phosphate PNA.Their triphosphate forms and particularly clofarabine triphosphatecompete with ATP for DNA synthesis, acts as proapoptotic agent and arepotent inhibitors of ribonucleotide reductase (RNR) which is involved intrinucleotide production.

Preferably, the inactivation of dCK in T cells is mediated by TALEnuclease. To achieve this goal, several pairs of dCK TALE-nuclease havebeen designed, assembled at the polynucleotide level and validated bysequencing. Examples of TALE-nuclease pairs which can be used accordingto the invention are depicted in PCT/EP2014/075317.

This dCK inactivation in T cells confers resistance to purine nucleosideanalogs (PNAs) such as clofarabine and fludarabine.

In a more preferred embodiment, the dCK inactivation in T cells iscombined with an inactivation of TRAC genes rendering these double knockout (KO) T cells both resistant to drug such as clofarabine and lessallogeneic than the same cell with an intact TCR. This double featuresis particularly useful for a therapeutic goal, allowing “off-the-shelf”allogeneic cells for immunotherapy in conjunction with chemotherapy totreat patients with cancer preferably refractory relapsed AML, or BPDNL.

This double KO inactivation dCK/TRAC can be performed simultaneously orsequentially before or after CD123 CAR of SEQ ID NO 31, of SEQ ID NO 32,or of SEQ ID NO 160 expression. One example of TALE-nuclease dCK/TRACpairs which gave success in the invention is described inPCT/EP2014/075317, in particular, the target sequences in the 2 loci(dCK and TRAC).

According to another aspect, the CD123 CAR expressing T-cell of theinvention (TCR KO anti-CD123 CAR of SEQ ID NO 31, of SEQ ID NO 32, or ofSEQ ID NO 160-expressing immune cells) can be further geneticallyengineered to improve its resistance to immunosuppressive drugs orchemotherapy treatments, which are used as standard care for treatingcancer.

Several cytotoxic agents (anti-cancer drugs) such as anti-metabolites,alkylating agents, anthracyclines, DNA methyltransferase inhibitors,platinum compounds and spindle poisons have been developed to killcancer cells. However, the introduction of these agents with noveltherapies, such as immunotherapies, is problematic as these drugs affectthe functioning/survival of immune T cells. For example, chemotherapyagents can be detrimental to the establishment of robust anti-tumorimmunocompetent cells due to the agents' non-specific toxicity profiles.Small molecule-based therapies targeting cell proliferation pathways mayalso hamper the establishment of anti-tumor immunity. If chemotherapyregimens that are transiently effective can be combined with novelimmunocompetent cell therapies then significant improvement inanti-neoplastic therapy might be achieved (for review (Dasgupta, McCartyet al. 2011).

To improve cancer therapy and selective engraftment of allogeneic TCRKO, CD123 CAR of SEQ ID NO 31, SEQ ID NO 32 or SEQ ID NO 160 expressingT-cells, drug resistance is conferred to said allogeneic T cells toprotect them from the toxic side effects of chemotherapy agent. The drugresistance of T-cells also permits their enrichment in or ex vivo, asT-cells which express the drug resistance gene will survive and multiplyrelative to drug sensitive cells.

Methods for engineering T-cells resistant to chemotherapeutic agents aredisclosed in PCT/EP2014/075317 which is fully incorporated by referenceherein.

In particular, the present invention discloses a method of engineeringallogeneic TCR KO, CD123 CAR of SEQ ID NO 31, SEQ ID NO 32 or SEQ ID NO160 expressing T-cells, suitable for immunotherapy wherein at least onegene encoding a T-cell receptor (TCR) component is inactivated and atleast one gene is modified to confer drug resistance, preferably the dCKgene is inactivated.

A method of engineering allogeneic CD123 CAR of SEQ ID NO 31, SEQ ID NO32 or SEQ ID NO 160 expressing T-cells suitable for combination therapywith purine analogues comprising:

-   -   Providing an anti-CD123 CAR of SEQ ID NO 31, SEQ ID NO 32, SEQ        ID NO 160 expressing T cell,    -   Modifying said anti-CD123 CAR expressing T-cell by inactivating        at least one gene encoding a T-cell receptor (TCR) component;    -   Modifying said anti-CD123 CAR expressing T-cell to confer drug        resistance to said anti-CD123 CAR expressing T-cell; preferably        to confer resistance to purine analogues    -   Expanding said engineered anti-CD123 CAR expressing T-cell in        the presence of said drug said drug is a purine analogue        selected from pentostatin, fludarabine 2-deoxyadenosine,        cladribine, clofarabine, Nelarabine, preferably pentostatin,        fludarabine monophosphate, and 2-chlorodeoxyadenosine (2-CDA).

Alternatively, the present invention relates to a method comprising:

-   -   Providing an anti-CD123 CAR of SEQ ID NO 31, of SEQ ID NO 32, or        of SEQ ID NO 160 expressing T cell,    -   Modifying said anti-CD123 CAR of SEQ ID NO 31, of SEQ ID NO 32,        or of SEQ ID NO 160 expressing T-cell to confer drug resistance        to said anti-CD123 CAR of SEQ ID NO 31, of SEQ ID NO 32, or of        SEQ ID NO 160 expressing T-cell; by deleting the dck gene,        preferably said drug is a purine analogue    -   Modifying said anti-CD123 CAR of SEQ ID NO 31, of SEQ ID NO 32,        or of SEQ ID NO 160 expressing T-cell by inactivating at least        one gene encoding a T-cell receptor (TCR) component;    -   Expanding said engineered anti-CD123 CAR of SEQ ID NO 31, of SEQ        ID NO 32, or of SEQ ID NO 160 expressing T-cell in the presence        of said drug, said drug is a purine analogue selected from        pentostatin, fludarabine 2-deoxyadenosine, cladribine,        clofarabine, Nelarabine, preferably pentostatin, fludarabine        monophosphate, and 2-chlorodeoxyadenosine (2-CDA).

In particular, the present invention relates to a method of engineeringallogeneic cells suitable for immunotherapy wherein a gene encoding aT-cell receptor (TCR alpha) component is inactivated and the dCK gene ismodified to confer resistance to purine analogues comprising:

-   -   Providing an anti-CD123 CAR expressing T-cell; in particular an        anti-CD123 CAR of SEQ ID NO 31, of SEQ ID NO 32, or of SEQ ID NO        160 expressing T cell,    -   Modifying said anti-CD123 CAR of SEQ ID NO 31, of SEQ ID NO 32,        or of SEQ ID NO 160 expressing T-cell by inactivating the gene        encoding the T-cell receptor (TCR alpha) component;    -   Inactivating the dCK gene in said anti-CD123 CAR of SEQ ID NO        31, of SEQ ID NO 32, or of SEQ ID NO 160 expressing T-cell to        confer resistance to purine analogues;    -   Expanding said engineered anti-CD123 CAR of SEQ ID NO 31, of SEQ        ID NO 32, or of SEQ ID NO 160 expressing T-cell in the presence        of said drug.

Alternatively, the present invention relates to a method comprising:

-   -   Providing an anti-CD123 CAR expressing T-cell; in particular an        anti-CD123 CAR of SEQ ID NO 31, of SEQ ID NO 32, or of SEQ ID NO        160 expressing T cell,    -   Inactivating the dCK gene in said anti-CD123 CAR of SEQ ID NO        31, of SEQ ID NO 32, or of SEQ ID NO 160 expressing T-cell to        confer resistance purine analogues    -   Modifying said anti-CD123 CAR of SEQ ID NO 31, of SEQ ID NO 32,        or of SEQ ID NO 160 CAR expressing T-cell by inactivating at        least one gene encoding a T-cell receptor (TCR alpha) component;    -   Expanding said engineered anti-CD123 CAR expressing T-cell in        the presence of purine analogues.

In a preferred embodiment dCK KO, TCR KO CD123 CAR of SEQ ID NO 31, ofSEQ ID NO 32, or of SEQ ID NO 160 CAR expressing T-cells provided areresistant to a drug selected from pentostatin, fludarabine2-deoxyadenosine, cladribine, clofarabine, or Nelarabine, preferably tofludarabine monophosphate, or to 2-chlorodeoxyadenosine (2-CDA).

Gene Expression Conferring Drug Resistance to Anti-CD123 CAR-ExpressingImmune Cells

In a particular embodiment, said drug resistance can be conferred to theT-cell of the invention by the expression of at least one drugresistance gene. Said drug resistance gene refers to a nucleic acidsequence that encodes “resistance” to an agent, such as achemotherapeutic agent (e.g. methotrexate). In other words, theexpression of the drug resistance gene in a cell permits proliferationof the cells in the presence of the agent to a greater extent than theproliferation of a corresponding cell without the drug resistance geneor survival in the presence of said drug. The expression of the drugresistance gene in a cell permits proliferation of the cells in thepresence of the agent and does not affect its activity. A drugresistance gene of the invention can encode resistance toanti-metabolite, methotrexate, vinblastine, cisplatin, alkylatingagents, anthracyclines, cytotoxic antibiotics, anti-immunophilins, theiranalogs or derivatives.

In one embodiment, a drug resistance gene confers resistance to a drug(or an agent), in particular an anti-cancer drug selected fromAracytine, Cytosine Arabinoside, amsacrine, Daunorubicine, Idarubicine,Novantrone, Mitoxantrone, Vepeside, Etoposide (VP16), arsenic trioxyde,transretinoic acid, combination of arsenic trioxyde, transretinoic acid,mechlorethamine, procarbazine, chlorambucil, cytarabine, anthracyclines,6-thioguanine, hydroxyurea, prednisone, and combination thereof.

Several drug resistance genes have been identified that can potentiallybe used to confer drug resistance the CD123 CAR expressing T cells ofthe invention (Takebe, Zhao et al. 2001; Sugimoto, Tsukahara et al.2003; Zielske, Reese et al. 2003; Nivens, Felder et al. 2004;Bardenheuer, Lehmberg et al. 2005; Kushman, Kabler et al. 2007).

One example of drug resistance gene can also be a mutant or modifiedform of Dihydrofolate reductase (DHFR) encoding gene. DHFR is an enzymeinvolved in regulating the amount of tetrahydrofolate in the cell and isessential to DNA synthesis. Folate analogs such as methotrexate (MTX)inhibit DHFR and are thus used as anti-neoplastic agents in clinic.Different mutant forms of DHFR which have increased resistance toinhibition by anti-folates used in therapy have been described. In aparticular embodiment, the drug resistance gene according to the presentinvention can be a nucleic acid sequence encoding a mutant form of humanwild type DHFR (GenBank: AAH71996.1) which comprises at least onemutation conferring resistance to an anti-folate treatment, such asmethotrexate. In particular embodiment, mutant form of DHFR comprises atleast one mutated amino acid at position G15, L22, F31 or F34,preferably at positions L22 or F31 (Schweitzer, Dicker et al. 1990);International application WO94/24277; U.S. Pat. No. 6,642,043). In aparticular embodiment, said DHFR mutant form comprises two mutated aminoacids at position L22 and F31. Correspondence of amino acid positionsdescribed herein is frequently expressed in terms of the positions ofthe amino acids of the form of wild-type DHFR polypeptide set forth inGenBank: AAH71996.1. In a particular embodiment, the serine residue atposition 15 is preferably replaced with a tryptophan residue. In anotherparticular embodiment, the leucine residue at position 22 is preferablyreplaced with an amino acid which will disrupt binding of the mutantDHFR to antifolates, preferably with uncharged amino acid residues suchas phenylalanine or tyrosine. In another particular embodiment, thephenylalanine residue at positions 31 or 34 is preferably replaced witha small hydrophilic amino acid such as alanine, serine or glycine.

As used herein, “antifolate agent” or “folate analogs” refers to amolecule directed to interfere with the folate metabolic pathway at somelevel. Examples of antifolate agents include, e.g., methotrexate (MTX);aminopterin; trimetrexate (Neutrexin™); edatrexate;N10-propargyl-5,8-dideazafolic acid (CB3717); ZD1694 (Tumodex),5,8-dideazaisofolic acid (IAHQ); 5,10-dideazatetrahydrofolic acid(DDATHF); 5-deazafolic acid; PT523 (N alpha-(4-amino-4-deoxypteroyl)-Ndelta-hemiphthaloyl-L-ornithine); 10-ethyl-10-deazaaminopterin (DDATHF,lomatrexol); piritrexim; 10-EDAM; ZD1694; GW1843; Pemetrexate and PDX(10-propargyl-10-deazaaminopterin).

Another example of drug resistance gene can also be a mutant or modifiedform of ionisine-5′-monophosphate dehydrogenase II (IMPDH2), arate-limiting enzyme in the de novo synthesis of guanosine nucleotides.The mutant or modified form of IMPDH2 is an IMPDH inhibitor resistancegene. IMPDH inhibitors can be mycophenolic acid (MPA) or its prodrugmycophenolate mofetil (MMF). The mutant IMPDH2 can comprises at leastone, preferably two mutations in the MAP binding site of the wild typehuman IMPDH2 (NP_000875.2) that lead to a significantly increasedresistance to IMPDH inhibitor. The mutations are preferably at positionsT333 and/or S351 (Yam, Jensen et al. 2006; Sangiolo, Lesnikova et al.2007; Jonnalagadda, Brown et al. 2013). In a particular embodiment, thethreonine residue at position 333 is replaced with an isoleucine residueand the serine residue at position 351 is replaced with a tyrosineresidue. Correspondence of amino acid positions described herein isfrequently expressed in terms of the positions of the amino acids of theform of wild-type human IMPDH2 polypeptide set forth in NP_000875.2.

Another drug resistance gene is the mutant form of calcineurin.Calcineurin (PP2B), an ubiquitously expressed serine/threonine proteinphosphatase that is involved in many biological processes and which iscentral to T-cell activation. Calcineurin is a heterodimer composed of acatalytic subunit (CnA; three isoforms) and a regulatory subunit (CnB;two isoforms). After engagement of the T-cell receptor, calcineurindephosphorylates the transcription factor NFAT, allowing it totranslocate to the nucleus and active key target gene such as IL2. FK506in complex with FKBP12, or cyclosporine A (CsA) in complex with CyPAblock NFAT access to calcineurin's active site, preventing itsdephosphorylation and thereby inhibiting T-cell activation (Brewin,Mancao et al. 2009). The drug resistance gene of the present inventioncan be a nucleic acid sequence encoding a mutant form of calcineurinresistant to calcineurin inhibitor such as FK506 and/or CsA. In aparticular embodiment, said mutant form can comprise at least onemutated amino acid of the wild type calcineurin heterodimer a atpositions: V314, Y341, M347, T351, W352, L354, K360, preferably doublemutations at positions T351 and L354 or V314 and Y341. In a particularembodiment, the valine residue at position 341 can be replaced with alysine or an arginine residue, the tyrosine residue at position 341 canbe replaced with a phenylalanine residue; the methionine at position 347can be replaced with the glutamic acid, arginine or tryptophane residue;the threonine at position 351 can be replaced with the glutamic acidresidue; the tryptophane residue at position 352 can be replaced with acysteine, glutamic acid or alanine residue, the serine at position 353can be replaced with the histidine or asparagines residue, the leucineat position 354 can be replaced with an alanine residue; the lysine atposition 360 can be replaced with an alanine or phenylalanine residue ofa sequence corresponding to GenBank: ACX34092.1. Correspondence of aminoacid positions described herein is frequently expressed in terms of thepositions of the amino acids of the form of wild-type human calcineurinheterodimer a polypeptide set forth in (GenBank: ACX34092.1).

In another particular embodiment, said mutant form can comprise at leastone mutated amino acid of the wild type calcineurin heterodimer b atpositions: V120, N123, L124 or K125, preferably double mutations atpositions L124 and K125. In a particular embodiment, the valine atposition 120 can be replaced with a serine, an aspartic acid,phenylalanine or leucine residue; the asparagine at position 123 can bereplaced with a tryptophan, lysine, phenylalanine, arginine, histidineor serine; the leucine at position 124 can be replaced with a threonineresidue; the lysine at position 125 can be replaced with an alanine, aglutamic acid, tryptophan, or two residues such as leucine-arginine orisoleucine-glutamic acid can be added after the lysine at position 125in the amino acid sequence corresponding to GenBank: ACX34095.1.Correspondence of amino acid positions described herein is frequentlyexpressed in terms of the positions of the amino acids of the form ofwild-type human calcineurin heterodimer b polypeptide set forth in(GenBank: ACX34095.1).

Another drug resistance gene is 0(6)-methylguanine methyltransferase(MGMT) encoding human alkyl guanine transferase (hAGT). AGT is a DNArepair protein that confers resistance to the cytotoxic effects ofalkylating agents, such as nitrosoureas and temozolomide (TMZ).6-benzylguanine (6-BG) is an inhibitor of AGT that potentiatesnitrosourea toxicity and is co-administered with TMZ to potentiate thecytotoxic effects of this agent. Several mutant forms of MGMT thatencode variants of AGT are highly resistant to inactivation by 6-BG, butretain their ability to repair DNA damage (Maze, Kurpad et al. 1999). Ina particular embodiment, AGT mutant form can comprise a mutated aminoacid of the wild type AGT position P140, in the amino acid sequence SEQID NO: 18 (UniProtKB: P16455). In a preferred embodiment, said prolineat position 140 is replaced with a lysine residue.

Another drug resistance gene can be multidrug resistance protein 1(MDR1) gene. This gene encodes a membrane glycoprotein, known asP-glycoprotein (P-GP) involved in the transport of metabolic byproductsacross the cell membrane. The P-Gp protein displays broad specificitytowards several structurally unrelated chemotherapy agents.

Overexpressing multidrug resistance protein 1 has been described toconfer resistance to drugs such as Mitoxantrone (Charles S. Morrow,Christina Peklak-Scott, Bimjhana Bishwokarma, Timothy E. Kute, Pamela K.Smitherman, and Alan J. Townsend. Multidrug Resistance Protein 1 (MRP1,ABCC1) Mediates Resistance to Mitoxantrone via Glutathione-DependentDrug Efflux Mol Pharmacol April 2006 69:1499-1505).

Thus, drug resistance can be conferred to cells by the expression ofnucleic acid sequence that encodes MDR-1 (NP_000918).

Still another way of preparing drug resistant cells according to theinvention is to prepare cells with specific mutation (s) such asmutations at Arg486 and Glu571 in the Human Topoisomerase II gene, toconfer resistance to amsacrine (S. PATEL, B. A. KELLER, and L. M.FISHER. 2000. MOLECULAR PHARMACOLOGY. Vol 57: p784-791 (2000).

Still another way of preparing drug resistant cells according to theinvention is to prepare cells overexpressing microRNA-21 to conferresistance to Daunorubicine (Involvement of miR-21 in resistance todaunorubicin by regulating PTEN expression in the leukaemia K562 cellline Bai, Haitao et al. FEBS Letters, Volume 585, Issue 2, 402-408).

In a preferred embodiment, cells of the invention are bearing such adrug resistance conferring mRNA or protein and also comprise aninhibitory mRNA or a gene, the expression of which is conditioned byanother drug, allowing the selective destruction of said drug resistantcells of the invention in the presence of said other drug or uponadministration of said other drug.

Drug resistance gene can also confer resistance to cytotoxicantibiotics, and can be ble gene or mcrA gene. Ectopic expression of blegene or mcrA in an immune cell gives a selective advantage when exposedto the chemotherapeutic agent, respectively the bleomycine or themitomycin C.

The most practical approach to gene therapy is the addition of a gene toengineer T-cell by using efficient gene delivery with vectors,preferably viral vector. Thus, in a particular embodiment, the presentinvention provides a method of conferring drug resistance to the CD123immune cells of the invention by introducing a transgene preferablyencoded by at least one vector into a cell.

The random insertion of genes into the genome may lead to theinappropriate expression of the inserted gene or the gene near theinsertion site. Specific gene therapy using homologous recombination ofexogenous nucleic acid comprising endogenous sequences to target genesto specific sites within the genome can allow a secure engineering ofT-cells. As described above, the genetic modification step of the methodaccording to the invention can comprise a step of introduction intocells of an exogeneous nucleic acid comprising at least a sequenceencoding the drug resistance gene and a portion of an endogenous genesuch that homologous recombination occurs between the endogenous geneand the exogeneous nucleic acid. In a particular embodiment, saidendogenous gene can be the wild type “drug resistance” gene, such thatafter homologous recombination, the wild type gene is replaced by themutant form of the gene which confers resistance to the drug.

Endonucleolytic breaks are known to stimulate the rate of homologousrecombination. Thus, in a particular embodiment, the method of theinvention further comprises the step of expressing in the cell arare-cutting endonuclease which is able to cleave a target sequencewithin an endogenous gene. Said endogenous gene can encode for examplesDHFR, IMPDH2, calcineurin or AGT. Said rare-cutting endonuclease can bea TALE-nuclease, a Zinc finger nuclease, a CRISPR/Cas9 endonuclease, aMBBBD-nuclease or a meganuclease.

Another example of enzyme which can be inactivated is humanhypoxanthine-guanine phosphoribosyl transferase (HPRT) gene (Genbank:M26434.1). In particular HPRT can be inactivated in engineered T-cellsto confer resistance to a cytostatic metabolite, the 6-thioguanine (6TG)which is converted by HPRT to cytotoxic thioguanine nucleotide and whichis currently used to treat patients with cancer, in particular leukemias(Hacke, Treger et al. 2013). Guanines analogs are metabolized by HPRTtransferase that catalyzes addition of phosphoribosyl moiety and enablesthe formation of TGMP Guanine analogues including 6 mercapthopurine(6MP) and 6 thioguanine (6TG) are usually used as lymphodepleting drugsto treat ALL. They are metabolized by HPRT (hypoxanthine phosphoribosyltransferase that catalyzes addition of phosphoribosyl moiety and enablesformation TGMP. Their subsequent phosphorylations lead to the formationof their triphosphorylated forms that are eventually integrated intoDNA. Once incorporated into DNA, thio GTP impairs fidelity of DNAreplication via its thiolate groupment and generate random pointmutation that are highly deleterious for cell integrity.

In another embodiment, the inactivation of the CD3 normally expressed atthe surface of the T-cell can confer resistance to anti-CD3 antibodiessuch as teplizumab.

Combination Treatment

The terms “therapeutic agent”, “chemotherapeutic agent”, or “drug” or“anti-cancer drug” as used herein refers to a medicament, preferably acompound or a derivative thereof that can interact with a cancer cell,thereby reducing the proliferative status of the cell and/or killing thecell. Examples of chemotherapeutic agents or “anti-cancer drug” include,but are not limited to, alkylating agents (e.g., Busulfan, Carboplatine,Chlorambucil, Cisplatine, Cyclophosphamide, Ifosfamide, Melphalan,Mechlorethamine, Oxaliplatine, Uramustine, •Temozolomide, Fotemustine),metabolic antagonists (e.g., purine nucleoside antimetabolite such asclofarabine, fludarabine or 2′-deoxyadenosine, methotrexate (MTX),5-fluorouracil or derivatives thereof, Azathioprine, Capecitabine,Cytarabine, •Floxuridine, •Fluorouracile, •Gemcitabine, •Methotrexate,Pemetrexed), antitumor antibiotics (e.g., mitomycin, Adriamycin,Bleomycine, •Daunorubicine, •Doxorubicine, •Epirubicine, •Hydroxyurea,•Idarubicine, •Mitomycin C, •Mitoxantrone), plant-derived antitumoragents (e.g., vincristine, vindesine, Taxol, Vinblastine,•(Vinorelbine), •Docetaxel, •Paclitaxel), topoisomerase inhibitor(Irinotecan, •Topotecan, •Etoposide).

In a preferred embodiment, a therapeutic agent, a chemotherapy drug asused herein refers to a compound or a derivative thereof that may beused to treat cancer, in particular to treat a hematopoietic cancer celland more particularly AML, and even more particular refractory relapsedAML thereby reducing the proliferative status of the cancer cell and/orkilling the cancer cell.

Other Examples of chemotherapeutic agents include, but are not limitedto Aracytine, Cytosine Arabinoside, Amsacrine, Daunorubicine,Idarubicine, Novantrone, Mitoxantrone, Vepeside, Etoposide (VP16),arsenic trioxyde, transretinoic acid, mechlorethamine, procarbazine,chlorambucil, and combination thereof.

In other embodiments of the present invention, cells of the inventionare administered to a patient in conjunction with a drug (or an agent)selected from Aracytine, Cytosine Arabinoside, amsacrine, Daunorubicine,Idarubicine, Novantrone, Mitoxantrone, Vepeside, Etoposide (VP16),arsenic trioxyde, transretinoic acid, cytarabine, anthracyclines,6-thioguanine, hydroxyurea, prednisone, and combination thereof.

Such agents may further include, but are not limited to, the anti-canceragents TRIMETHOTRIXATE™ (TMTX), TEMOZOLOMIDE™, RALTRITREXED™,S-(4-Nitrobenzyl)-6-thioinosine (NBMPR), 6-benzyguanidine (6-BG),bis-chloronitrosourea (BCNU) and CAMPTOTHECIN™, or a therapeuticderivative of any thereof.

In a more preferred embodiment an anti-CD123 CAR of SEQ ID NO 31, of SEQID NO 32, or of SEQ ID NO 160 expressing T cell with a TCR and a dck KOgene, is administered to a patient, in combination with at least onetherapeutic agent selected from Aracytine, Cytosine Arabinoside,Amsacrine, Daunorubicine, Idarubicine, Novantrone, Mitoxantrone,Vepeside, Etoposide (VP16), arsenic trioxyde, transretinoic acid andcombination thereof.

An anti-CD123 CAR of SEQ ID NO 31, of SEQ ID NO 32, or of SEQ ID NO 160expressing T cell with a TCR and a dck KO gene, in combination with atleast one therapeutic agent selected from Aracytine, CytosineArabinoside, Amsacrine, Daunorubicine, Idarubicine, Novantrone,Mitoxantrone, Vepeside, Etoposide (VP16), arsenic trioxyde,transretinoic acid and combination thereof is used as a therapeuticagent.

Preferably, an anti-CD123 CAR of SEQ ID NO 31, of SEQ ID NO 32, or ofSEQ ID NO 160 expressing T cell with a TCR and a dck KO gene, incombination with a purine analogue (fludarabine), is used as atherapeutic agent.

Preferably, an anti-CD123 CAR of SEQ ID NO 31, of SEQ ID NO 32, or ofSEQ ID NO 160 expressing T cell with a TCR and a dck KO gene, incombination with a FLAG treatment, is used as a therapeutic agent.

In one embodiment, said CD123 CAR is selected from SEQ ID NO 76 to SEQID NO. 117 and expressed in a double TCR dCK KO T cells and combined toa purine analogue or to FLAG as a therapeutic agent.

In the present invention a combination treatment comprises infusion of amAb for sorting and/or depletion purpose(s). In a preferred embodiment,said mAb is Rixutimab.

The maximum dose of Rituximab to be administered preferably byintravenous route is 2,250 mg/m2. It is no administered as anintravenous push or bolus.

Multiple Drug Resistance of Anti-CD123 CAR-Expressing Immune Cells

In another particular embodiment, the inventors sought to develop an“off-the shelf” immunotherapy strategy, using allogeneic T-cells, inparticular allogenic anti-CD123 CAR expressing T-cell resistant tomultiple drugs to mediate selection of engineered T-cells when thepatient is treated with different drugs. The therapeutic efficiency canbe significantly enhanced by genetically engineering multiple drugresistance allogeneic T-cells. Such a strategy can be particularlyeffective in treating tumors that respond to drug combinations thatexhibit synergistic effects. Moreover multiple resistant engineeredT-cells can expand and be selected using minimal dose of drug agents.

Thus, the method according to the present invention can comprisemodifying T-cell of the invention to confer multiple drug resistance tosaid T-cell of the invention. Said multiple drug resistance can beconferred by either expressing more than one drug resistance gene or byinactivating more than one drug sensitizing gene. In another particularembodiment, the multiple drug resistance can be conferred to said T-cellby expressing at least one drug resistance gene and inactivating atleast one drug sensitizing gene. In particular, the multiple drugresistance can be conferred to said T-cell by expressing at least onedrug resistance gene such as mutant form of DHFR, mutant form of IMPDH2,mutant form of calcineurin, mutant form of MGMT, the ble gene, and themcrA gene and inactivating at least one drug sensitizing gene such asHPRT gene. In a preferred embodiment, multiple drug resistance can beconferred by inactivating HPRT gene and expressing a mutant form ofDHFR; or by inactivating HPRT gene and expressing a mutant form ofIMPDH2; or by inactivating HPRT gene and expressing a mutant form ofcalcineurin; by inactivating HPRT gene and expressing a mutant form ofMGMT; by inactivating HPRT gene and expressing the ble gene; byinactivating HPRT gene and expressing the mcrA gene.

In one embodiment, the present invention provides allogenic anti-CD123CAR expressing T-cell expressing more than one drug resistance gene orwherein more than one drug sensitizing gene is inactivated.

Suicide Genes in Anti-CD123 CAR-Expressing Immune Cells of the Invention

In some instances, since engineered T-cells can expand and persist foryears after administration, it is desirable to include a safetymechanism to allow selective deletion of administrated T-cells. Thus, insome embodiments, the method of the invention comprises thetransformation of said T-cells with a recombinant suicide gene. Saidrecombinant suicide gene is used to reduce the risk of direct toxicityand/or uncontrolled proliferation of said T-cells once administrated ina subject (Quintarelli C, Vera F, blood 2007; Tey S K, Dotti G., RooneyC M, boil blood marrow transplant 2007). Suicide genes enable selectivedeletion of transformed cells in vivo. In particular, the suicide genehas the ability to convert a non-toxic pro-drug into cytotoxic drug orto express the toxic gene expression product. In other words, “Suicidegene” is a nucleic acid coding for a product, wherein the product causescell death by itself or in the presence of other compounds.

A representative example of such a suicide gene is one which codes forthymidine kinase of herpes simplex virus. Additional examples arethymidine kinase of varicella zoster virus and the bacterial genecytosine deaminase which can convert 5-fluorocytosine to the highlytoxic compound 5-fluorouracil. Suicide genes also include as nonlimiting examples caspase-9 or caspase-8 or cytosine deaminase.Caspase-9 can be activated using a specific chemical inducer ofdimerization (CID). Suicide genes can also be polypeptides that areexpressed at the surface of the cell and can make the cells sensitive totherapeutic monoclonal antibodies. As used herein “prodrug” means anycompound useful in the methods of the present invention that can beconverted to a toxic product. The prodrug is converted to a toxicproduct by the gene product of the suicide gene in the method of thepresent invention. A representative example of such a prodrug isganciclovir which is converted in vivo to a toxic compound byHSV-thymidine kinase. The ganciclovir derivative subsequently is toxicto tumor cells. Other representative examples of prodrugs includeacyclovir, FIAU[1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodouracil],6-methoxypurine arabinoside for VZV-TK, and 5-fluorocytosine forcytosine deaminase.

One preferred suicide gene system of the invention employs a recombinantantigenic polypeptide comprising antigenic motif recognized by theanti-CD20 mAb Rituximab, especially QBen10, such as in the so-calledRQR8 polypeptide described in WO2013153391. Rituximab, an authorizedantibody drug, can then be used for cell depletion when needed.

In one embodiment, the present invention provides a TCR KO-dCK KOanti-CD123 CAR of SEQ ID NO. 31 or 32 expressing T-cell, expressing aRQR8 suicide gene allowing said cells to be selectively destroyed.

More preferably, the present invention provides a TCR KO-dCK KOanti-CD123 CAR expressing T-cell, wherein said CD123 CAR comprises asuicide domain allowing CD123 CAR expressing cells to be selectivelydestroyed, preferably said suicide domain comprised at least two domainsof SEQ ID NO 161, more preferably said CD123 CAR comprises a sequence ofSEQ ID NO 171.

CD123+/Luc+ Drug Resistant Daudi Cells for Testing the Cytotoxicity ofDrug Resistant Allogenic CART Cells

The present invention encompasses also a method for manufacturing targetcells which express both CD123 and are resistant to purine analogues.These target cells are particularly useful for testing the cytotoxicityof CD123 CAR T cells of the invention. These cells are readily resistantto clinically relevant dose of clofarabine and harbor luciferaseactivity. This combination of features enable traking them in vivo in amice model or destroy them when required.

More particularly, they can be used to assess the cytotoxicityproperties drug resistant T cells in mice in the presence of clofarabineor other PNAs. Clofarabine resistant Daudi cells mimick thephysiological state of acute lymphoblastic leukemia (ALL) patientsrelapsing form induction therapy, that harbor drug resistant B cellmalignancies. Thus, these cells are of great interest to evaluate thereliability and cytotoxicity of drug resistant CAR T cells. Preferably,these target cells are CD123+ Luciferase+ Daudi cells.

Isolated Cell

The present invention relates to an isolated cell expressing a CD123 CARwhich binds to CD123. Thus, the invention relates to an anti-CD123 CARexpressing cell. In a particular embodiment, said anti-CD123 CARexpressing cell is resistant to at least one drug and/or comprises atleast one disrupted gene encoding a T-cell receptor component.

In a preferred embodiment, the present invention relates to an isolatedT cell expressing a CAR which binds to CD123 and is resistant to atleast one purine analogue and comprises a disrupted gene encoding aT-cell receptor alpha component.

In a more preferred embodiment, the present invention relates to anisolated T cell expressing a CAR of SEQ ID NO 31, 32 or 160 which bindsto CD123 and destroys CD123 expressing cells and is resistant to atleast one purine analogue and comprises a disrupted gene encoding aT-cell receptor alpha component.

In a more preferred embodiment, the present invention relates to anisolated T cell expressing a CAR of SEQ ID NO 160 which binds to CD123and destroys CD123 expressing cells and is resistant to at least onepurine analogue and comprises a disrupted gene encoding a T-cellreceptor alpha component.

In a more preferred embodiment, the present invention relates to anisolated T cell expressing a CAR of SEQ ID NO 160 which binds to CD123and destroys CD123 expressing cells and is resistant to at least onepurine analogue and comprises a disrupted gene encoding a T-cellreceptor beta component.

In another particular embodiment, said anti-CD123 CAR expressing T cellcomprises at least one disrupted drug sensitizing gene such as dCK orHPRT gene. In a more particular embodiment, said isolated anti-CD123 CART-cell comprises a disrupted HPRT gene and express a DHFR mutant; saidisolated anti-CD123 CAR T-cell comprises a disrupted HPRT gene andexpress a IMPDH2 mutant; said isolated anti-CD123 CAR T-cell comprises adisrupted HPRT gene and express a calcineurin mutant; said isolatedanti-CD123 CAR T-cell comprises a disrupted HPRT gene and express a AGTmutant.

In particular, the present invention relates to an isolated T-cell, inparticular an isolated allogeneic anti-CD123 CAR expressing T-cell, andpreferably an isolated allogeneic anti-CD123 CAR expressing T-cellcomprising a peptide having a sequence of SEQ ID NO. 31 32 or 160, saidisolated allogeneic anti-CD123 CAR expressing T-cell is moreparticularly resistant to a purine analogue, and specifically suitablefor immunotherapy in the presence of purine analogues.

In one aspect, the present invention provides methods for engineering anisolated immune cells to make it resistant to purine analogs (purinenucleotide analogs or PNA), such a clorofarabine or fludarabine, so thatthey can be used in cancer immunotherapy treatments in patientspre-treated with these conventional chemotherapies.

The resistance to drugs can be conferred to the isolated T-cell of theinvention by inactivating one or more gene(s) responsible for the cell'ssensitivity to the drug (drug sensitizing gene(s)), such as the dcKand/or HPRT genes.

Immune Check Points

The present invention provides allogeneic dCK KO T-cells expressing ananti-CD123 CAR, in particular an anti-CD123 CAR of SEQ ID No 31, or ofSEQ ID No 32, or of SEQ ID No 160 wherein at least one gene expressingone or more component of T-cell receptor (TCR) is inactivated and/or onegene selected from the genes CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22,PDCD1, LAG3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7,SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6,CASP7, FADD, FAS, TGFBRII, TGFBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI,SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, CSK, PAG1, SIT1, FOXP3,PRDM1 (orblimp1), BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, isinactivated as referred to in WO2014/184741.

According to further aspect of the invention, the immune cells can befurther manipulated to make them more active or limit exhaustion, byinactivating genes encoding proteins that act as “immune checkpoints”that act as regulators of T-cells activation, such as the following geneselected from CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, LAG3, HAVCR2,BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244,TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS,TGFBRII, TGFBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA,IL10RB, HMOX2, IL6R, IL6ST, CSK, PAG1, SIT1, FOXP3, PRDM1 (orblimp1),BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, preferably, said gene is PDCD1or CTLA-4. Examples of genes, which expression could be reduced orsuppressed in T cells of the invention are indicated in Table 9.

In one embodiment said gene is a gene that acts as a regulator ofT-cells activation coding the beta 2 microglobulin protein.

According to a further aspect of the invention, the anti-CD123CAR-immune cells of the invention can be further manipulated to makethem resistant to a drug, in particular to a drug used duringchemotherapy against cancer, in particular a CD123-expressingcell-mediated cancer such as AML. This can be achieved by introducing agene conferring resistance to said drug. This same gene may be turned onand off by using a gene inducible inhibition/expression system aspreviously described (Garcia E L, Mills A A (2002) Getting aroundlethality with inducible Cre-mediated excision. Semin Cell Dev Biol13:151-8, Lewandoski M (2001) Conditional control of gene expression inthe mouse. Nat Rev Genet 2:743-55; Scharfenberger L, Hennerici T, KirlyG et al. (2014) Transgenic mouse technology in skin biology: Generationof complete or tissue-specific knockout mice. J Invest Dermatol 134:e16;Schwenk F, Kuhn R, Angrand P O et al. (1998) Temporally and spatiallyregulated somatic mutagenesis in mice. Nucleic Acids Res 26:1427-32.

Thus, anti-CD123 CAR of sequence corresponding to SEQ ID NO 31, to SEQID NO 32 or to SEQ ID NO 160-expressing, PNA resistant immune T cell,wherein (i) at least one gene expressing one or more component of T-cellreceptor (TCR) is inactivated (ii) at least one gene conferringresistance to a drug (other than PNA) is incorporated or a geneconferring sensitivity to said drug (other than PNA) is deleted ormutated to be inactivated (iii) another gene selected from the genedisclosed in table 9 is inactivated—is an object of the presentinvention.

The present invention encompasses the isolated anti-CD123 CAR-immunecells or cell lines obtainable by the method of the invention, moreparticularly isolated cells comprising any of the proteins,polypeptides, allelic variants, altered or deleted genes or vectorsdescribed herein.

This object is provided for the treatment of a cancer, in particularAML, refractory relapse AML, BPDNL.

The immune cells of the present invention or cell lines can furthercomprise exogenous recombinant polynucleotides, in particular CARs orsuicide gene(s) (encoding SEQ ID NO. 161) or they can comprise alteredor deleted genes coding for checkpoint proteins or ligands thereof thatcontribute to their efficiency as a therapeutic product, ideally as an“off the shelf” product.

In another aspect, the present invention concerns the method fortreating or preventing cancer in the patient by administrating at leastonce an engineered immune cell obtainable by the above methods.

TABLE 5 List of genes encoding immune checkpoint proteins modified inengineered T cells of the invention Genes that can be inactivatedPathway In the pathway Co-inhibitory CTLA4 (CD152) CTLA4, PPP2CA,PPP2CB, receptors PTPN6, PTPN22 (prefered) PDCD1 (PD-1, PDCD1 (preferedand more CD279) prefered) CD223 (lag3) LAG3 HAVCR2 (tim3) HAVCR2BTLA(cd272) BTLA CD160(by55) CD160 IgSF family TIGIT CD96 CRTAMLAIR1(cd305) LAIR1 SIGLECs SIGLEC7 SIGLEC9 CD244(2b4) CD244 Death TRAILTNFRSF10B, TNFRSF10A, receptors CASP8, CASP10, CASP3, CASP6, CASP7 FASFADD, FAS Cytokine TGF-beta signaling TGFBRII, TGFBRI, SMAD2, signallingSMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1 IL10 signalling IL10RA, IL10RB,HMOX2 IL6 signalling IL6R, IL6ST Prevention CSK, PAG1 of TCR SIT1signalling Induced Treg induced Treg FOXP3 Transcription transcriptionfactors PRDM1 (=blimp1, heterozygotes factors controlling mice controlchronic viral controlling exhaustion infection better than wt orexhaustion conditional KO) Stat 5 BATF Hypoxia iNOS induced GUCY1A2,GUCY1A3, mediated guanylated cyclase GUCY1B2, GUCY1B3 tolerance

One, preferably two alleles, of above genes are modified in engineered Tcells of the invention the locus is verified for each preparation ofTALEN used to KO said gene/allele.

In a preferred embodiment said method of further engineer the immunecells involves introducing into said T cells polynucleotides, inparticular mRNAs, encoding specific rare-cutting endonuclease toselectively inactivate the genes mentioned above by DNA cleavage. In amore preferred embodiment said rare-cutting endonucleases areTALE-nucleases or Cas9 endonuclease. TAL-nucleases have so far provenhigher specificity and cleavage efficiency over the other types ofrare-cutting endonucleases, making them the endonucleases of choice forproducing of the engineered immune cells on a large scale with aconstant turn-over.

Delivery Methods

The different methods described above involve expressing a protein ofinterest such as drug resistance gene, rare-cutting endonuclease,Chimeric Antigen Receptor (CAR), in particular an anti-CD123 CAR andmore particularly, a CAR comprising a SEQ ID NO. 31, or 32 or 160and/including a suicide domain into an isolated cell.

As non-limiting example, said protein of interest can be expressed inthe cell by its introduction as a transgene preferably encoded by atleast one plasmid vector. Polypeptides may be expressed in the cell as aresult of the introduction of polynucleotides encoding said polypeptidesinto the cell. Alternatively, said polypeptides could be producedoutside the cell and then introduced thereto.

Methods for introducing a polynucleotide construct into cells are knownin the art and include as non limiting examples stable transformationmethods wherein the polynucleotide construct is integrated into thegenome of the cell, transient transformation methods wherein thepolynucleotide construct is not integrated into the genome of the celland virus mediated methods.

Said polynucleotides may be introduced into a cell by for example,recombinant viral vectors (e.g. retroviruses, adenoviruses), liposomeand the like. For example, transient transformation methods include forexample microinjection, electroporation or particle bombardment, cellfusion. Said polynucleotides may be included in vectors, moreparticularly plasmids or virus, in view of being expressed in cells.Said plasmid vector can comprise a selection marker which provides foridentification and/or selection of cells which received said vector.

Different transgenes can be included in one vector. Said vector cancomprise a nucleic acid sequence encoding ribosomal skip sequence suchas a sequence encoding a 2A peptide. 2A peptides, which were identifiedin the Aphthovirus subgroup of picornaviruses, causes a ribosomal “skip”from one codon to the next without the formation of a peptide bondbetween the two amino acids encoded by the codons (see Donnelly et al.,J. of General Virology 82: 1013-1025 (2001); Donnelly et al., J. of Gen.Virology 78: 13-21 (1997); Doronina et al., Mol. And. Cell. Biology28(13): 4227-4239 (2008); Atkins et al., RNA 13: 803-810 (2007)).

By “codon” is meant three nucleotides on an mRNA (or on the sense strandof a DNA molecule) that are translated by a ribosome into one amino acidresidue. Thus, two polypeptides can be synthesized from a single,contiguous open reading frame within an mRNA when the polypeptides areseparated by a 2A oligopeptide sequence that is in frame. Such ribosomalskip mechanisms are well known in the art and are known to be used byseveral vectors for the expression of several proteins encoded by asingle messenger RNA.

In a more preferred embodiment of the invention, polynucleotidesencoding polypeptides according to the present invention can be mRNAwhich is introduced directly into the cells, for example byelectroporation. The inventors determined the optimal condition for mRNAelectroporation in T-cell. The inventor used the cytoPulse technologywhich allows, by the use of pulsed electric fields, to transientlypermeabilize living cells for delivery of material into the cells. Thetechnology, based on the use of PulseAgile (BTX Havard Apparatus, 84October Hill Road, Holliston, Mass. 01746, USA) electroporationwaveforms grants the precise control of pulse duration, intensity aswell as the interval between pulses (U.S. Pat. No. 6,010,613 andInternational PCT application WO2004083379). All these parameters can bemodified in order to reach the best conditions for high transfectionefficiency with minimal mortality. Basically, the first high electricfield pulses allow pore formation, while subsequent lower electric fieldpulses allow moving the polynucleotide into the cell.

The different methods described above involve introducing a CD123 CAR ofSEQ ID NO. 31, SEQ ID NO 32 or SEQ ID NO 160 into a cell. Asnon-limiting example, said CAR can be introduced as transgenes encodedby one plasmid vector. Said plasmid vector can also contain a selectionmarker which provides for identification and/or selection of cells whichreceived said vector.

Polypeptides may be synthesized in situ in the cell as a result of theintroduction of polynucleotides encoding said polypeptides into thecell. Alternatively, said polypeptides could be produced outside thecell and then introduced thereto. Methods for introducing apolynucleotide construct into cells are known in the art and includingas non limiting examples stable transformation methods wherein thepolynucleotide construct is integrated into the genome of the cell,transient transformation methods wherein the polynucleotide construct isnot integrated into the genome of the cell and virus mediated methods.Said polynucleotides may be introduced into a cell by for example,recombinant viral vectors (e.g. retroviruses, adenoviruses), liposomeand the like. For example, transient transformation methods include forexample microinjection, electroporation or particle bombardment. Saidpolynucleotides may be included in vectors, more particularly plasmidsor virus, in view of being expressed in cells.

Engineered Immune Cells

The present invention also relates to isolated cells or cell linessusceptible to be obtained by said method to engineer cells. Inparticular said isolated cell comprises at least one CAR as describedabove. In another embodiment, said isolated cell comprises a populationof CARs each one comprising different extracellular ligand bindingdomains. In particular, said isolated cell comprises exogenouspolynucleotide sequence encoding CAR. Genetically modified immune cellsof the present invention are activated and proliferate independently ofantigen binding mechanisms.

In the scope of the present invention is also encompassed an isolatedimmune cell, preferably a T-cell obtained according to any one of themethods previously described. Said immune cell refers to a cell ofhematopoietic origin functionally involved in the initiation and/orexecution of innate and/or adaptative immune response. Said immune cellaccording to the present invention can be derived from a stem cell. Thestem cells can be adult stem cells, non-human embryonic stem cells, moreparticularly non-human stem cells, cord blood stem cells, progenitorcells, bone marrow stem cells, induced pluripotent stem cells,totipotent stem cells or hematopoietic stem cells. Representative humancells are CD34+ cells. Said isolated cell can also be a dendritic cell,killer dendritic cell, a mast cell, a NK-cell, a B-cell or a T-cellselected from the group consisting of inflammatory T-lymphocytes,cytotoxic T-lymphocytes, regulatory T-lymphocytes or helperT-lymphocytes. In another embodiment, said cell can be derived from thegroup consisting of CD4+T-lymphocytes and CD8+T-lymphocytes. Prior toexpansion and genetic modification of the cells of the invention, asource of cells can be obtained from a subject through a variety ofnon-limiting methods. Cells can be obtained from a number ofnon-limiting sources, including peripheral blood mononuclear cells, bonemarrow, lymph node tissue, cord blood, thymus tissue, tissue from a siteof infection, ascites, pleural effusion, spleen tissue, and tumors. Incertain embodiments of the present invention, any number of T cell linesavailable and known to those skilled in the art, may be used. In anotherembodiment, said cell can be derived from a healthy donor, from apatient diagnosed with cancer or from a patient diagnosed with aninfection. In another embodiment, said cell is part of a mixedpopulation of cells which present different phenotypic characteristics.In the scope of the present invention is also encompassed a cell lineobtained from a transformed T-cell according to the method previouslydescribed. Modified cells resistant to an immunosuppressive treatmentand susceptible to be obtained by the previous method are encompassed inthe scope of the present invention.

As a preferred embodiment, the present invention provides T-cells or apopulation of T-cells endowed with a CD123 CAR having a sequencecorresponding to SEQ ID NO. 31, SEQ ID NO. 32 or SEQ ID NO 160 asdescribed above, that do not express functional TCR are resistant to PNAand that a reactive towards CD123 positive cells, for their adoptivetransfer into patients, preferably into patients suffering AML,refractory relapse AML, BPDNL or a lymphoproliferative disorder, or as atreatment before bone marrow transplantation.

As a more preferred embodiment, the present invention provides T-cellsor a population of T-cells endowed with a CD123 CAR CD123 CAR having asequence corresponding to SEQ ID NO. 31, SEQ ID NO. 32 or SEQ ID NO 160and that a reactive towards CD123 positive cells as described above,that do not express a functional TCR and are resistant to a selectedPNA, for their allogeneic transplantation into patients treated withsaid selected PNA.

In an even more preferred embodiment, the present invention providesT-cells or a population of TRC KO dCK KO T-cells endowed with ananti-CD123 CAR comprising a polypeptide of CD123 CAR having a sequencecorresponding to SEQ ID NO. 31, SEQ ID NO. 32 or SEQ ID NO 160 incombination with another treatment.

Activation and Expansion of T Cells

Whether prior to or after genetic modification of the T cells, even ifthe genetically modified immune cells of the present invention areactivated and proliferate independently of antigen binding mechanisms,the immune cells, particularly T-cells of the present invention can befurther activated and expanded generally using methods as described, forexample, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964;5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869;7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; andU.S. Patent Application Publication No. 20060121005. T cells can beexpanded in vitro or in vivo.

Generally, the T cells of the invention are expanded by contact with anagent that stimulates a CD3 TCR complex and a co-stimulatory molecule onthe surface of the T cells to create an activation signal for theT-cell. For example, chemicals such as calcium ionophore A23187, phorbol12-myristate 13-acetate (PMA), or mitogenic lectins likephytohemagglutinin (PHA) can be used to create an activation signal forthe T-cell.

As non-limiting examples, T cell populations may be stimulated in vitrosuch as by contact with an anti-CD3 antibody, or antigen-bindingfragment thereof, or an anti-CD2 antibody immobilized on a surface, orby contact with a protein kinase C activator (e.g., bryostatin) inconjunction with a calcium ionophore. For co-stimulation of an accessorymolecule on the surface of the T cells, a ligand that binds theaccessory molecule is used. For example, a population of T cells can becontacted with an anti-CD3 antibody and an anti-CD28 antibody, underconditions appropriate for stimulating proliferation of the T cells.Conditions appropriate for T cell culture include an appropriate media(e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 5, (Lonza))that may contain factors necessary for proliferation and viability,including serum (e.g., fetal bovine or human serum), interleukin-2(IL-2), insulin, IFN-g, 1L-4, 1L-7, GM-CSF, -10, -2, 1L-15, TGFp, andTNF- or any other additives for the growth of cells known to the skilledartisan. Other additives for the growth of cells include, but are notlimited to, surfactant, plasmanate, and reducing agents such asN-acetyl-cysteine and 2-mercaptoethanoi. Media can include RPMI 1640,A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1, and X-Vivo 20, Optimizer, withadded amino acids, sodium pyruvate, and vitamins, either serum-free orsupplemented with an appropriate amount of serum (or plasma) or adefined set of hormones, and/or an amount of cytokine(s) sufficient forthe growth and expansion of T cells. Antibiotics, e.g., penicillin andstreptomycin, are included only in experimental cultures, not incultures of cells that are to be infused into a subject. The targetcells are maintained under conditions necessary to support growth, forexample, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g.,air plus 5% CO2). T cells that have been exposed to varied stimulationtimes may exhibit different characteristics

In another particular embodiment, said cells can be expanded byco-culturing with tissue or cells. Said cells can also be expanded invivo, for example in the subject's blood after administrating said cellinto the subject.

Pharmaceutical Compositions

Provided herein are pharmaceutical compositions comprising thegenetically engineered immune cells of the invention, e.g., geneticallyengineered TCR KO-dCK KO CD123 CAR T cells having a sequencecorresponding to SEQ ID NO. 31, SEQ ID NO. 32 or SEQ ID NO 160.

Provided herein are pharmaceutical compositions comprising thegenetically engineered immune cells of the invention, e.g., geneticallyengineered TCR KO-dCK KO CD123 CAR T cells having a sequencecorresponding to any one of the sequences selected from SEQ ID NO 188 toSEQ ID NO 197.

In another embodiment, pharmaceutical compositions comprising thegenetically engineered immune T cells of the invention, e.g.,genetically engineered TCR KO dCK KO CD123 CAR having any one of thesequence selected from having a sequence selected from SEQ ID NO. 34 toSEQ ID NO. 159, preferably from SEQ ID NO. 34, SEQ ID NO. 76, SEQ ID NO.36, SEQ ID NO. 78; SEQ ID NO. 37, SEQ ID NO. 79, SEQ ID NO. 41, SEQ IDNO. 83, SEQ ID NO. 42, SEQ ID NO. 8), SEQ ID NO. 43, SEQ ID NO. 85, SEQID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 88,SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 52, SEQ ID NO.53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 94, SEQ ID NO. 95, SEQ IDNO. 96, and SEQ ID NO. 97, T cells are provided.

In accordance with this disclosure, the term “pharmaceuticalcomposition” relates to a composition for administration to anindividual. In a preferred embodiment, the pharmaceutical compositioncomprises a composition for parenteral, transdermal, intraluminal,intra-arterial, intrathecal or intravenous administration (iv) or fordirect injection into a cancer. It is in particular envisaged that saidpharmaceutical composition is administered to the individual viainfusion or injection iv. Administration of the suitable compositionsmay be effected by different ways, e.g., by intravenous (iv),subcutaneous, intraperitoneal, intramuscular, topical or intradermaladministration.

The pharmaceutical composition of the present disclosure may furthercomprise a pharmaceutically acceptable carrier. Examples of suitablepharmaceutical carriers are well known in the art and include phosphatebuffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions, etc.Compositions comprising such carriers can be formulated by well-knownconventional methods. These pharmaceutical compositions can beadministered to the subject at a suitable dose.

The dosage regimen will be determined by the attending physician andclinical factors. As is well known in the medical arts, dosages for anyone patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. An example of a dosage for administrationmight be in the range of 0.24 μg to 48 mg of cells according to theinvention, preferably 0.24 μg to 24 mg, more preferably 0.24 μg to 2.4mg, even more preferably 0.24 μg to 1.2 mg and most preferably 0.24 μgto 240 mg units per kilogram of body weight per day. Particularlypreferred dosages are recited herein below. Progress can be monitored byperiodic assessment.

The CAR cell compositions of the disclosure may be administered locallyor systemically. Administration will generally be parenteral, e.g.,intravenous; DNA may also be administered directly to the target site,e.g., by biolistic delivery to an internal or external target site or bycatheter to a site in an artery. In a preferred embodiment, thepharmaceutical composition is administered subcutaneously and in an evenmore preferred embodiment intravenously. Preparations for parenteraladministration include sterile aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. Intravenous vehicles include fluid andnutrient replenishes, electrolyte replenishers (such as those based onRinger's dextrose), and the like. Preservatives and other additives mayalso be present such as, for example, antimicrobials, anti-oxidants,chelating agents, and inert gases and the like. In addition, thepharmaceutical composition of the present disclosure might compriseproteinaceous carriers, like, e.g., serum albumin or immunoglobulin,preferably of human origin. It is envisaged that the pharmaceuticalcomposition of the disclosure might comprise, in addition to theproteinaceous bispecific single chain antibody constructs or nucleicacid molecules or vectors encoding the same (as described in thisdisclosure), further biologically active agents, depending on theintended use of the pharmaceutical composition.

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, one or more cells according to the invention foruse in cell therapy and/or the reagents to generate one or more cellsfor use in cell therapy that harbors recombinant expression vectors maybe comprised in a kit. The kit components are provided in suitablecontainer means.

Some components of the kits may be packaged either in aqueous media orin lyophilized form and under frozen packages. The container means ofthe kits will generally include at least one vial, test tube, flask,bottle, syringe or other container means, into which a component may beplaced, and preferably, suitably aliquoted. Where there are more thanone component in the kit, the kit also will generally contain a second,third or other additional container into which the additional componentsmay be separately placed. However, various combinations of componentsmay be comprised in a vial. The kits also will typically include a meansfor containing the components in close confinement for commercial sale.Such containers may include injection or blow molded plastic containersinto which the desired vials are retained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly useful. In some cases, the containermeans may itself be a syringe, pipette, and/or other such likeapparatus, from which the formulation may be applied to an infected areaof the body, injected into an animal, and/or even applied to and/ormixed with the other components of the kit.

In a preferred embodiment one and/or more liquid solutions iscryoresistant.

However, the components of the kit or part of it may be provided asdried powder(s) or as a tablet. When reagents and/or components areprovided as a dry powder, the powder can be reconstituted by theaddition of a suitable solvent. It is envisioned that the solvent mayalso be provided in another container means. The kits may also comprisea second container means for containing a sterile, pharmaceuticallyacceptable buffer and/or other diluent.

In particular embodiments, cells that are to be used for cell therapyare provided in a kit, and in some cases the cells are essentially thesole component of the kit. The kit may comprise reagents and materialsto make the desired cell. In specific embodiments, the reagents andmaterials include primers for amplifying desired sequences, nucleotides,suitable buffers or buffer reagents, salt, and so forth, and in somecases the reagents include vectors and/or DNA that encodes a CARmolecule as described herein and/or regulatory elements therefor.

In particular embodiments, there are one or more apparatuses in the kitsuitable for extracting one or more samples from an individual. Theapparatus may be a syringe, scalpel, and so forth.

In some cases, the kit, in addition to cell therapy embodiments, alsoincludes a second cancer therapy, such as chemotherapy, hormone therapy,and/or immunotherapy, for example. The kit(s) may be tailored to aparticular cancer for an individual and comprise respective secondcancer therapies for the individual. Preferably, said other cancertherapy is a purine analogue, a FLAG treatment.

Therapeutic uses of engineered T-cells Comprising a CD123 CAR of theinvention.

In various embodiments CAR constructs, nucleic acid sequences, vectors,host cells, as contemplated herein and/or pharmaceutical compositionscomprising the same are used for the prevention, treatment oramelioration of a cancerous disease, such as a tumorous disease. Inparticular embodiments, the pharmaceutical composition of the presentdisclosure may be particularly useful in preventing, ameliorating and/ortreating cancer, including cancer having solid tumors, for example.

In particular embodiments, provided herein are a method of treating anindividual for cancer, comprising the step of providing atherapeutically effective amount of a plurality of any of cells of thedisclosure to the individual.

In certain aspects, the cancer is a solid tumor, and the tumor may be ofany size, but in specific embodiments, the solid tumors are about 2 mmor greater in diameter. In certain aspects, the method further comprisesthe step of providing a therapeutically effective amount of anadditional cancer therapy to the individual.

As used herein “treatment” or “treating,” includes any beneficial ordesirable effect on the symptoms or pathology of a disease orpathological condition, and may include even minimal reductions in oneor more measurable markers of the disease or condition being treated,e.g., cancer. Treatment can involve optionally either the reduction oramelioration of symptoms of the disease or condition, or the delaying ofthe progression of the disease or condition. “Treatment” does notnecessarily indicate complete eradication or cure of the disease orcondition, or associated symptoms thereof.

As used herein, “prevent,” and similar words such as “prevented,”“preventing” etc., indicate an approach for preventing, inhibiting, orreducing the likelihood of the occurrence or recurrence of, a disease orcondition, e.g., cancer. It also refers to delaying the onset orrecurrence of a disease or condition or delaying the occurrence orrecurrence of the symptoms of a disease or condition. As used herein,“prevention” and similar words also includes reducing the intensity,effect, symptoms and/or burden of a disease or condition prior to onsetor recurrence of the disease or condition.

In particular embodiments, the present invention contemplates, in part,cells, CAR constructs, nucleic acid molecules and vectors that canadministered either alone or in any combination using standard vectorsand/or gene delivery systems, and in at least some aspects, togetherwith a pharmaceutically acceptable carrier or excipient. In certainembodiments, subsequent to administration, said nucleic acid moleculesor vectors may be stably integrated into the genome of the subject.

In specific embodiments, viral vectors may be used that are specific forcertain cells or tissues and persist in said cells. Suitablepharmaceutical carriers and excipients are well known in the art. Thecompositions prepared according to the disclosure can be used for theprevention or treatment or delaying the above identified diseases.

Furthermore, the disclosure relates to a method for the prevention,treatment or amelioration of a tumorous disease comprising the step ofadministering to a subject or individual in the need thereof aneffective amount of immune cells, e.g., T cells or cytotoxic Tlymphocytes, harboring a CD123 CAR of SEQ ID NO. 31 or a CD123 CAR ofSEQ ID NO. 32 or a CD123 CAR of SEQ ID NO. 160; a nucleic acid sequenceencoding a the same; a vector comprising a nucleotide sequence encodingsaid CD123 CAR, as described herein and/or produced by a process asdescribed herein.

Possible indications for administration of the composition(s) of theexemplary CD123 CAR cells are cancerous diseases, including tumorousdiseases, including:

The administration of the composition(s) of the disclosure is useful forall stages and types of lymphoproliferative disorder, including forminimal residual disease, early cancer, advanced cancer, and/or relapsedand/or refractory cancer.

The disclosure further encompasses co-administration protocols withother compounds, e.g. bispecific antibody constructs, targeted toxins orother compounds, which act via immune cells.

The clinical regimen for co-administration of the inventive compound(s)may encompass co-administration at the same time, before or after theadministration of the other component. Particular combination therapiesinclude chemotherapy, radiation, surgery, hormone therapy, or othertypes of immunotherapy.

Particular doses for therapy may be determined using routine methods inthe art. However, in specific embodiments, the engineered CD123 T cellsof the invention are delivered to an individual in need thereof once,although in some cases it is multiple times, including 2, 3, 4, 5, 6, ormore times. When multiple doses are given, the span of time betweendoses may be of any suitable time, but in specific embodiments, it isdays or weeks or months between the doses. Doses and origins of the Tcell donor are selected to avoid any undesirable side effects. The timebetween doses may vary in a single regimen and may depend on therecipient (patient in need thereof). In particular embodiments, the timebetween doses is 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days or weeks. Inspecific cases, it is between 4-8 or 6-8 weeks, for example.

In specific embodiments, one regimen includes one of the following doseregimen

of CD123 T cell of the invention:11×10⁴/m²,11×10⁵/m²,11×10⁶/m²,23×10⁶/m²,31×10⁷/m² 43×10⁷/m²,51×10⁸/m²,or from 10⁴ to from 10¹⁰ cells/kg.

In an alternative embodiment, the T cells are provided to the individualin the following dose regimen:

Dose Level CD123 CTL Dose

11×10⁶/m²,21×10⁷/m²,31×10⁸/m²,6 10⁴ to from 5·10⁸ cells/kg,

An efficient amount of the CD123 CAR engineered immune cell of theinvention corresponds to such dose that may be adapted depending on thestatus of each treated individual.

Therapeutic Applications

In another embodiment, isolated cell(s) obtained by the differentmethods or cell derived (after one to 50 in vitro passages) from saidisolated cell of the invention can be used as a medicament. Geneticallyengineered TCR KO dCK KO CD123 CART cells of the invention that can beused as a medicament can be those having a CD123 CAR having a sequencecorresponding to any one of SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO 160,SEQ ID NO. 34, to SEQ ID NO. 159.

In a preferred embodiment a genetically engineered TCR KO dCK KO CD123CAR T cells having a sequence corresponding to SEQ ID NO. 31, SEQ ID NO.32 or SEQ ID NO 160. is provided for use as a medicament.

In another preferred embodiment, a, genetically engineered TCR KO dCK KOCD123 CAR T cells having any one of the sequence selected from having asequence selected from SEQ ID NO. 34, SEQ ID NO. 76, SEQ ID NO. 36, SEQID NO. 78, SEQ ID NO. 37, SEQ ID NO. 79, SEQ ID NO. 41, SEQ ID NO. 83,SEQ ID NO. 42, SEQ ID NO. 8), SEQ ID NO. 43, SEQ ID NO. 85, SEQ ID NO.46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 88, SEQ IDNO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 52, SEQ ID NO. 53, SEQID NO. 54, SEQ ID NO. 55, SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96,SEQ ID NO. 97, is provided for use as a medicament.

In another preferred embodiment, a genetically engineered TCR KO dCK KOCD123 CAR T cells having any one of the sequence selected from SEQ IDNO. 160, SEQ ID NO. 188, SEQ ID NO. 189, SEQ ID NO. 190, SEQ ID NO. 191;SEQ ID NO. 192, SEQ ID NO. 193, SEQ ID NO. 194, SEQ ID NO. 195, SEQ IDNO. 196, SEQ ID NO. 197 is provided for use as a medicament.

In another embodiment, said medicament can be used for treating cancer,particularly for the treatment of B-cell lymphomas and leukemia in apatient in need thereof.

In another embodiment, said isolated cell according to the invention orcell line derived from said isolated cell can be used in the manufactureof a medicament for treatment of a cancer in a patient in need thereof.

In a particular embodiment, an anti-CD123 CAR expressing T cell isprovided as a medicament for the treatment of AML, of an AML subtype, ofan AML-related complication, of an AML-related condition. In a preferredembodiment, an anti-CD123 CAR expressing T cell wherein said anti-CD123CAR comprises SEQ ID NO 31, SEQ ID NO 32 or SEQ ID NO 160 is provided asa medicament.

In another embodiment, said medicament can be used for treating aCD123-expressing cell-mediated pathological condition or a conditioncharacterized by the direct or indirect activity of a CD123-expressingcell. In other words, the invention is related to an anti-CD123 CARexpressing T cell comprising 80% to 100% of SEQ ID NO 31, SEQ ID NO 32or SEQ ID NO 160 for its use as a medicament to treat a condition linkedto the detrimental activity of CD123-expressing cells, in particular totreat a condition selected from AML, AML subtype, AML-relatedcomplication, and AML-related conditions;

In another aspect, the present invention relies on methods for treatingpatients in need thereof, said method comprising at least one of thefollowing steps:

-   -   (a) providing an immune-cell obtainable by any one of the        methods previously described;    -   (b) Administrating said transformed immune cells to said        patient,

On one embodiment, said T cells of the invention can undergo robust invivo T cell expansion and can persist for an extended amount of time.

In a preferred embodiment said T cell is destroyed by administration ofa drug or an antibody that selectively destroys said T cells.

In a more preferred embodiment genetically engineered TCR KO dCK KOCD123 CAR T cells of the invention having a CD123 CAR having a sequencecorresponding to any one of SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO 160,SEQ ID NO. 34, to SEQ ID NO. 159 are uses as a medicament/a treatmentand then cleared up from the patient using an antibody recognizing thesuicide domain RQR8, or CD20 domain, or SEQ ID NO 161—using preferablyrituximab.

Said treatment can be ameliorating, curative or prophylactic. It may beeither part of an autologous immunotherapy or part of an allogenicimmunotherapy treatment. By autologous, it is meant that cells, cellline or population of cells used for treating patients are originatingfrom said patient or from a Human Leucocyte Antigen (HLA) compatibledonor. By allogeneic is meant that the cells or population of cells usedfor treating patients are not originating from said patient but from adonor.

Cells of the invention that can be used with the disclosed methods aredescribed in the previous section. Said treatment can be used to treatpatients diagnosed wherein a pre-malignant or malignant cancer conditioncharacterized by CD123-expressing cells, especially by an overabundanceof CD123-expressing cells. Such conditions are found in hematologiccancers, such as leukemia or malignant lymphoproliferative disorders.Lymphoproliferative disorder can be lymphoma, in particular multiplemyeloma, non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicularlymphoma (small cell and large cell).

Cancers that may be treated using the cells of the invention or apharmaceutical composition comprising said cell may comprise nonsolidtumors (such as hematological tumors, including but not limited to pre-BALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuselarge B-cell lymphoma, BPDNL, AML, refractory relapse AML, or beforebone marrow transplantation.

Types of cancers to be treated with the CD123 cell expressing CARs ofthe invention include, but are not limited leukemia or lymphoidmalignancies. Adult tumors/cancers and pediatric tumors/cancers are alsoincluded.

In one embodiment, the present invention provides a pharmaceuticalcomposition for its use in the treatment of a CD123 expressingcells-mediated disease, in particular a CD123 expressing cells—mediatedhematologic cancer, said composition comprising said anti-CD123 CARexpressing T cell of the invention, preferably said anti-CD123 CAR is ofSEQ ID NO 31, SEQ ID NO 32 or SEQ ID NO 160, more preferably having asequence selected from SEQ ID NO. 160, SEQ ID NO. 188, SEQ ID NO. 189,SEQ ID NO. 190, SEQ ID NO. 191; SEQ ID NO. 192, SEQ ID NO. 193, SEQ IDNO. 194, SEQ ID NO. 195, SEQ ID NO. 196, SEQ ID NO. 197 Any otherCD123-mediating or CD123-involving malignant lymphoproliferativedisorders disclosed herein may be improved with the anti-CD123CAR-expressing cells of the present invention.

In a preferred embodiment, the cancer that may be treated using theanti-CD123 CAR-expressing cells of the present invention is leukemia(AML), a disease associated to leukemia or a complication thereof.

Leukemias that can be treated using the anti-CD123 CAR-expressing cellsof the present invention can be acute myelogenous leukemia (AML),chronic myelogenous leukemia, melodysplastic syndrome, acute lymphoidleukemia, chronic lymphoid leukemia, and myelodysplastic syndrome.

AML or AML subtypes that may be treated using the anti-CD123CAR-expressing cells of the present invention may be in particular,acute myeloblastic leukemia, minimally differentiated acute myeloblasticleukemia, acute myeloblastic leukemia without maturation, acutemyeloblastic leukemia with granulocytic maturation, promyelocytic oracute promyelocytic leukemia (APL), acute myelomonocytic leukemia,myelomonocytic together with bone marrow eosinophilia, acute monoblasticleukemia (M5a) or acute monocytic leukemia (M5b), acute erythroidleukemias, including erythroleukemia (M6a) and very rare pure erythroidleukemia (M6b), acute megakaryoblastic leukemia, acute basophilicleukemia, acute panmyelosis with myelofibrosis, whether involvingCD123-positive cells.

Subtypes of AML that may be treated using the anti-CD123 CAR-expressingcells of the present invention also include, hairy cell leukemia,philadelphia chromosome-positive acute lymphoblastic leukemia.

AML may be classified as AML with specific genetic abnormalities.Classification is based on the ability of karyotype to predict responseto induction therapy, relapse risk, survival.

Accordingly, AML that may be treated using the anti-CD123 CAR-expressingcells of the present invention of SEQ ID NO 31, SEQ ID NO 32 or SEQ IDNO 160 may be AML with a translocation between chromosomes 8 and 21, AMLwith a translocation or inversion in chromosome 16, AML with atranslocation between chromosomes 9 and 11, APL (M3) with atranslocation between chromosomes 15 and 17, AML with a translocationbetween chromosomes 6 and 9, AML with a translocation or inversion inchromosome 3, AML (megakaryoblastic) with a translocation betweenchromosomes 1 and 22.

The present invention is particularly useful for the treatment of AMLassociated with these particular cytogenetic markers.

The present invention also provides an anti-CD123 CAR expressing T cellof SEQ ID NO 31, SEQ ID NO 32 or SEQ ID NO 160 for the treatment ofpatients with specific cytogenetic subsets of AML, such as patients witht(15; 17)(q22; q21) identified using all-trans retinoic acid (ATRA)16-19and for the treatment of patients with t(8; 21)(q22; q22) orinv(16)(p13q22)/t(16; 16)(p13; q22) identified using repetitive doses ofhigh-dose cytarabine.

Preferably, the present invention provides an anti-CD123 CAR expressingT cell of SEQ ID NO 31, SEQ ID NO 32 or SEQ ID NO 160 for the treatmentof patients with aberrations, such as −5/del(5q), −7, abnormalities of3q, or a complex karyotype, who have been shown to have inferiorcomplete remission rates and survival, in combination with a FLAG.

Group of Patients

In a preferred embodiment, the invention provides an anti-CD123 CARexpressing T cell of SEQ ID NO 31, SEQ ID NO 32 or SEQ ID NO 160 as atreatment for AML in patients over 60 years or in patients of less than20 years.

In a more preferred embodiment, the present invention provides ananti-CD123 CAR expressing T cell of SEQ ID NO 31, SEQ ID NO 32 or SEQ IDNO 160 for use as a pediatric treatment, in particular a pediatrictreatment against AML, or AML-related diseases or complications.

In still another preferred embodiment, the present invention is used asa treatment in AML patients with low, poor or unfavorable status that isto say with a predicted survival of less than 5 years survival rate. Inthis group, patients suffering AML with the following cytogeneticcharacteristics: −5; 5q; −7; 7q-; 11q23; non t(9; 11); inv(3); t(3; 3);t(6; 9); t(9; 22) is associated with poor-risk status (Byrd J. C. etal., Dec. 15, 2002; Blood: 100 (13) and is especially contemplated to betreated according to the present invention or with an object of thepresent invention.

In one embodiment, the anti-CD123 CAR expressing T cell of presentinvention may be used as induction therapy, as post remission therapy ofAML or as a consolidation therapy in patient with AML. Preferably, TRCKO cells or TCR KO and dck KO T cells expressing an anti-CD123 CAR ofSEQ ID NO. 31, SEQ ID NO. 32 or SEQ ID NO. 160 are used as postremission therapy of AML or as a consolidation therapy in patient withAML.

In one embodiment, the TCR KO, dck KO, anti-CD123 CAR of SEQ ID NO 31,SEQ ID NO 32 or SEQ ID NO 160 expressing T cell of the present inventionmay be used in case of AML relapse, or in case of refractory orresistant AML. Preferably, said TCR KO T dck KO, anti-CD123 CAR of SEQID NO 31, SEQ ID NO 32 or SEQ ID NO 160 expressing T cell of the presentinvention are used in patients with AML relapse, or with refractory orresistant AML, more preferably, in combination with at least one otheranti-cancer drug

In another preferred embodiment, TCR KO, dck KO, anti-CD123 CAR of SEQID NO 31, SEQ ID NO 32 or SEQ ID NO 160 expressing T cell of the presentinvention is used for preventing cancer cells development occurring inparticular after anti-cancer treatment, during bone marrow depletion orbefore bone marrow transplantation, after bone marrow destruction.

AML Complications

In one particular embodiment the invention provides a medicament thatimproves the health condition of a patient, in particular a patientundergoing a complication related to AML. More preferably, said TCR KO,dck KO, anti-CD123 CAR of SEQ ID NO 31, SEQ ID NO 32 or SEQ ID NO 160expressing T cell is used as a medicament for the treatment of acomplication related to AML, preferably with FLAG.

A complication or disease related to AML may include a precedingmyelodysplasia phase, secondary leukemia, in particular secondary AML,high white blood cell count, and absence of Auer rods. Among others,leukostasis and involvement of the central nervous system (CNS),Hyperleukocytosis, residual disease, are also considered as acomplication or disease related to AML.

AML Associated Diseases

In one embodiment, the present invention also provides a TCR KO, dck KO,anti-CD123 CAR of SEQ ID NO 31, SEQ ID NO 32 or SEQ ID NO 160 expressingT cell for the treatment of a pathological condition related to AML.Preferably, the present invention provides a TCR KO, dck KO, anti-CD123CAR of SEQ ID NO 31, SEQ ID NO 32 or SEQ ID NO 160 expressing T cell forthe treatment of a pathological condition related to AML.

The present invention provides a therapy (a TCR KO, dck KO, anti-CD123CAR of SEQ ID NO 31, SEQ ID NO 32 or SEQ ID NO 160 expressing T cell,preferably having a sequence selected from SEQ ID NO. 160, SEQ ID NO.188, SEQ ID NO. 189, SEQ ID NO. 190, SEQ ID NO. 191; SEQ ID NO. 192, SEQID NO. 193, SEQ ID NO. 194, SEQ ID NO. 195, SEQ ID NO. 196, SEQ ID NO.197) for AML related myeloid neoplasms, for acute myeloid leukemia andmyelodysplastic syndrome, a treatment of relapsed or refractory acutemyeloid leukemia, a treatment of relapsed or refractory acutepromyelocytic leukemia in adults, a treatment for acute promyeloidleukaemia, a treatment of acute myeloid leukemia in adults over 60years.

According to another aspect, the present invention provides acomposition for the treatment of AML associated diseases, in particularhematologic malignancy related to AML.

Hematologic malignancy related to AML conditions include myelodysplasiasyndromes (MDS, formerly known as “preleukemia”) which are a diversecollection of hematological conditions united by ineffective production(or dysplasia) of myeloid blood cells and risk of transformation to AM

In another embodiment, the invention provides a medicament that improvesthe health state of a patient suffering multiple myeloma.

Other pathological conditions or genetic syndromes associated with therisk of AML can be improved with the adequate use of the presentinvention, said genetic syndromes include Down syndrome, trisomy,Fanconi anemia, Bloom syndrome, Ataxia-telangiectasia, Diamond-Blackfananemia, Schwachman-Diamond syndrome, Li-Fraumeni syndrome,Neurofibromatosis type 1, Severe congenital neutropenia (also calledKostmann syndrome)

Other CD123-Mediated Pathological Conditions

According to another aspect, the present invention provides acomposition for the treatment of CD123+ cell-mediated diseases. TheseCD123+ cell mediated diseases include inflammation, autoimmune diseases.

In particular, the present invention can be used for the treatment ofCD123+ cell mediated diseases such as inflammation of thegastrointestinal mucosae and more particularly, inflammatory boweldiseases, nasal allergy, inflammation of the skin such as juveniledermatomyositis, hematodermia.

The present invention can be used as a medicament for the treatment ofCD123+ cell mediated diseases such as autoimmune diseases in particularKikushi disease.

Preferably, the present invention provides a treatment for a recurrentinfection including infection due to viruses such as Epstein-Barr virus,herpes simplex virus, in particular oncogenic viruses, HHV-8, HHV-6,HTLV or HIV, parasitic infection such as infection due to plasmodiumfalciparum, Plasmodium vivax, Plasmodium ovale, or Plasmodium malariae.

In particular, the present invention provides a treatment forEpstein-Barr virus lymphadenitis, herpes simplex virus lymphadenitis.

In another aspect, the present invention provides a composition for thetreatment of systemic lupus erythematosus lymphadenitis, tuberculosis,cystic fibrosis, hepatitis, biliary atresia, in particular virus-inducedhepatitis or biliary atresia in children, autoimmune hepatitis; primarybiliary cirrhosis.

Composition Comprising an Engineered T Cells According to the Inventionfor Use as a Medicament and Method

The present invention also provides a composition comprising agenetically engineered immune TCR KO-dCK KO-CD123 CAR having a sequencecorresponding to SEQ ID NO. 31, SEQ ID NO. 32 or SEQ ID NO 160 T cells,or endowed with a sequence selected from SEQ ID NO. 160, SEQ ID NO. 188,SEQ ID NO. 189, SEQ ID NO. 190, SEQ ID NO. 191; SEQ ID NO. 192, SEQ IDNO. 193, SEQ ID NO. 194, SEQ ID NO. 195, SEQ ID NO. 196, SEQ ID NO. 197or

a genetically engineered immune TCR KO-dCK KO CD123 CAR having any oneof the sequence selected from SEQ ID NO. 34 to SEQ ID NO. 159,preferably from SEQ ID NO. 34, SEQ ID NO. 76, SEQ ID NO. 36, SEQ ID NO.78; SEQ ID NO. 37, SEQ ID NO. 79, SEQ ID NO. 41, SEQ ID NO. 83, SEQ IDNO. 42, SEQ ID NO. 8), SEQ ID NO. 43, SEQ ID NO. 85, SEQ ID NO. 46, SEQID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 88, SEQ ID NO. 89,SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO.54, SEQ ID NO. 55, SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96 and SEQID NO. 97, T cells.

Preferably, the present invention provides a composition comprising agenetically engineered immune TCR KO-dCK KO-CD123 CAR having a sequencecorresponding to SEQ ID NO. 31 T cells.

Preferably, the present invention provides a composition comprising agenetically engineered immune TCR KO-dCK KO-CD123 CAR having a sequencecorresponding to SEQ ID NO. 32 T cells.

Preferably, the present invention provides a composition comprising agenetically engineered immune TCR KO-dCK KO-CD123 CAR having a sequencecorresponding to SEQ ID NO. 160 T cells

Or any one of the sequence selected from SEQ ID NO. 160, SEQ ID NO. 188,SEQ ID NO. 189, SEQ ID NO. 190, SEQ ID NO. 191; SEQ ID NO. 192, SEQ IDNO. 193, SEQ ID NO. 194, SEQ ID NO. 195, SEQ ID NO. 196, SEQ ID NO. 197,

for its use or a method for treating a cancer such as BPDNL or as atreatment before bone marrow transplant. In one aspect, the disease is ahematologic cancer, in particular a stem cell cancer including but isnot limited to leukemia (such as acute myelogenous leukemia (AML),chronic myelogenous leukemia, acute lymphoid leukemia, chronic lymphoidleukemia and myelodysplasia syndrome) and malignant lymphoproliferativeconditions, including lymphoma (such as multiple myeloma, non-Hodgkin'slymphoma, Burkitt's lymphoma, and small cell- and large cell-follicularlymphoma), or a complication (relapse refractor AML), thereof.

The present invention also provides a composition as above for its useor a method for inhibiting the proliferation or reducing aCD123-expressing cell population or activity in a patient. An exemplarymethod includes contacting a population of cells comprising aCD123-expressing cell with a CD 123 CART cell of the invention thatbinds to the CD123-expressing cell.

In a more specific aspect, the present invention provides a compositionfor its use or a method for inhibiting the proliferation or reducing thepopulation of cancer cells expressing CD 123 in a patient, the methodscomprising contacting the CD123-expressing cancer cell population with aCD 123 CART cell of the invention that binds to the CD123-expressingcell, binding of a CD 123 CART cell of the invention to theCD123-expressing cancer cell resulting in the destruction of theCD123-expressing cancer cells

In certain aspects, the CD 123 CART cell of the invention reduces thequantity, number, amount or percentage of cells and/or cancer cells byat least 25%, at least 30%, at least 40%, at least 50%, at least 65%, atleast 75%, at least 85%, at least 95%, or at least 99% (to undetectablelevel) in a subject with or animal model for myeloid leukemia or anothercancer associated with CD123-expressing cells, relative to a negativecontrol.

The present invention also provides a composition for its use or amethod for preventing, treating and/or managing a disorder or conditionassociated with CD123-expressing cells (e.g., associated with ahematologic cancer), the methods comprising administering to a subjectin need a CD 123 CART cell of the invention that binds to theCD123-expressing cell. In one aspect, the subject is a human.Non-limiting examples of disorders associated with CD123-expressingcells include autoimmune disorders (such as lupus), inflammatorydisorders (such as allergies, IBD, and asthma) and cancers (such ashematological cancers, in particular AML or AML complications).

The present invention also provides a composition for its use or amethod for preventing, treating and/or managing a disease associatedwith CD123-expressing cells, the method comprising administering to asubject in need a CD 123 CART cell of the invention that binds to theCD123-expressing cell. In one aspect, the subject is a human.Non-limiting examples of diseases associated with CD123-expressing cellsinclude Acute Myeloid Leukemia (AML), myelodysplasia, B-cell AcuteLymphoid Leukemia, T-cell Acute Lymphoid Leukemia, hairy cell leukemia,blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myeloidleukemia, Hodgkin lymphoma.

The present invention provides a composition for its use or a method fortreating or preventing relapse of cancer associated withCD123-expressing cells, the method comprising administering to a subjectin need thereof a CD 123 CART cell of the invention that binds to the CD123-expressing cell. In another aspect, the methods compriseadministering to the subject in need thereof an effective amount of a CD123 CART cell of the invention that binds to the CD123-expressing cellin combination with an effective amount of another therapy.

In one aspect, CD 123 is considered to be a “cancer stem cell” marker inAML. Therefore, a CD 123 CART cell of the invention can prevent relapseof AML, or even treat AML that is mostly CD 123-negative but with a“stem” population of CD 123+ cells (a CD123-expressing cells).

In one aspect, the invention provides compositions and methods fortreating subjects that have undergone treatment for a disease ordisorder associated with elevated expression levels of CD 19, andexhibits a disease or disorder associated with elevated levels of CD123.

In one aspect, B-cell acute lymphoid leukemia (ALL) is an example ofdisease requiring a serial treatment using CART cells. For example,treatment with anti-CD 19 CAR T cells can sometimes result inCD19-negative relapse, which can be treated with anti-CD123 CAR T cellsof the invention. Alternatively, the present invention includes dualtargeting of B-ALL using CART cells comprising an anti-CD 19 CAR and ananti-CD 123 CAR.

The treatment with the engineered immune cells according to theinvention may be in combination with one or more therapies againstcancer selected from the group of antibodies therapy, chemotherapy,cytokines therapy, dendritic cell therapy, gene therapy, hormonetherapy, laser light therapy and radiation therapy.

Preferably, the treatment with the engineered immune cells according tothe invention may be administered in combination (e.g., before,simultaneously or following) with one or more therapies against cancerselected from Aracytine, Cytosine Arabinoside, amsacrine, Daunorubicine,Idarubicine, Novantrone, Mitoxantrone, Vepeside, Etoposide (VP16),arsenic trioxyde, transretinoic acid, combination of arsenic trioxyde,transretinoic acid, mechlorethamine, procarbazine, chlorambucil, andcombination thereof.

According to a preferred embodiment of the invention, said treatment canbe administrated into patients undergoing an immunosuppressivetreatment. Indeed, the present invention preferably relies on cells orpopulation of cells, which have been made resistant to at least oneimmunosuppressive agent due to the inactivation of a gene encoding areceptor for such immunosuppressive agent. In this aspect, theimmunosuppressive treatment should help the selection and expansion ofthe T-cells according to the invention within the patient.

The administration of the cells or population of cells according to thepresent invention may be carried out in any convenient manner, includingby aerosol inhalation, injection, ingestion, transfusion, implantationor transplantation. The compositions described herein may beadministered to a patient subcutaneously, intradermaly, intratumorally,intranodally, intramedullary, intramuscularly, by intravenous orintralymphatic injection, or intraperitoneally. In one embodiment, thecell compositions of the present invention are preferably administeredby intravenous injection.

The administration of the cells or population of cells can consist ofthe administration of from 10⁴-10⁹ cells per kg body weight, preferably10⁵ to 10⁶ cells/kg body weight including all integer values of cellnumbers within those ranges. The cells or population of cells can beadministrated in one or more doses. In another embodiment, saideffective amount of cells are administrated as a single dose. In anotherembodiment, said effective amount of cells are administrated as morethan one dose over a period time. Timing of administration is within thejudgment of managing physician and depends on the clinical condition ofthe patient. The cells or population of cells may be obtained from anysource, such as a blood bank or a donor. While individual needs vary,determination of optimal ranges of effective amounts of a given celltype for a particular disease or conditions within the skill of the art.An effective amount means an amount which provides a therapeutic orprophylactic benefit. The dosage administrated will be dependent uponthe age, health and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment and the nature of the effectdesired.

In another embodiment, said effective amount of cells or compositioncomprising those cells are administrated parenterally. Saidadministration can be an intravenous administration. Said administrationcan be directly done by injection within a tumor.

In certain embodiments of the present invention, cells are administeredto a patient in conjunction with (e.g., before, simultaneously orfollowing) any number of relevant treatment modalities, including butnot limited to treatment with agents such as antiviral therapy,cidofovir and interleukin-2, Cytarabine (also known as ARA-C) ornatalizimab treatment for MS patients or efaliztimab treatment forpsoriasis patients or other treatments for PML patients. In furtherembodiments, the T cells of the invention may be used in combinationwith chemotherapy, radiation, immunosuppressive agents, such ascyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunoablative agents such as CAMPATH, anti-CD3antibodies or other antibody therapies, cytoxin, fludaribine,cyclosporin, FK506, rapamycin, mycoplienolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin) (Henderson, Naya et al. 1991; Liu, Albers et al. 1992;Bierer, Hollander et al. 1993). In a further embodiment, the cellcompositions of the present invention are administered to a patient inconjunction with (e.g., before, simultaneously or following) bone marrowtransplantation, T cell ablative therapy using either chemotherapyagents such as, fludarabine, external-beam radiation therapy (XRT),cyclophosphamide, or antibodies such as OKT3 or CAMPATH, In anotherembodiment, the cell compositions of the present invention areadministered following B-cell ablative therapy such as agents that reactwith CD20, e.g., Rituxan. In that case, the CD123 CAR expressed in Tcell of the invention does not consist in SEQ ID 160, unless it isadministered before Rituxan.

For example, in one embodiment, subjects may undergo standard treatmentwith high dose chemotherapy followed by peripheral blood stem celltransplantation. In certain embodiments, following the transplant,subjects receive an infusion of the expanded immune cells of the presentinvention. In an additional embodiment, expanded cells are administeredbefore or following surgery.

In certain embodiments of the present invention, anti-CD123 CARexpressing cells are administered to a patient in conjunction (e.g.,before, simultaneously or following) with a drug selected fromAracytine, Cytosine Arabinoside, amsacrine, Daunorubicine, Idarubicine,Novantrone, Mitoxantrone, Vepeside, Etoposide (VP16), arsenic trioxyde,transretinoic acid, mechlorethamine, procarbazine, chlorambucil, andcombination thereof. In these embodiments anti-CD123 CAR expressingcells may be resistant to the particular drug or combination of drugsthat is (are) administered in conjunction with anti-CD123 CAR expressingcells.

In other embodiments of the present invention, anti-CD123 CAR expressingcells are administered to a patient in conjunction with a drug selectedfrom cytarabine, anthracyclines, 6-thioguanine, hydroxyurea, prednisone,and combination thereof.

Other Definitions

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one. Amino acid residuesin a polypeptide sequence are designated herein according to theone-letter code, in which, for example, Q means Gln or Glutamineresidue, R means Arg or Arginine residue and D means Asp or Asparticacid residue.

Amino acid substitution means the replacement of one amino acid residuewith another, for instance the replacement of an Arginine residue with aGlutamine residue in a peptide sequence is an amino acid substitution.

Nucleotides are designated as follows: one-letter code is used fordesignating the base of a nucleoside: an is adenine, t is thymine, c iscytosine, and g is guanine. For the degenerated nucleotides, rrepresents g or a (purine nucleotides), k represents g or t, srepresents g or c, w represents a or t, m represents a or c, yrepresents t or c (pyrimidine nucleotides), d represents g, a or t, vrepresents g, a or c, b represents g, t or c, h represents a, t or c,and n represents g, a, t or c.

“As used herein, “nucleic acid” or “polynucleotides” refers tonucleotides and/or polynucleotides, such as deoxyribonucleic acid (DNA)or ribonucleic acid (RNA), oligonucleotides, fragments generated by thepolymerase chain reaction (PCR), and fragments generated by any ofligation, scission, endonuclease action, and exonuclease action. Nucleicacid molecules can be composed of monomers that are naturally-occurringnucleotides (such as DNA and RNA), or analogs of naturally-occurringnucleotides (e.g., enantiomeric forms of naturally-occurringnucleotides), or a combination of both. Modified nucleotides can havealterations in sugar moieties and/or in pyrimidine or purine basemoieties. Sugar modifications include, for example, replacement of oneor more hydroxyl groups with halogens, alkyl groups, amines, and azidogroups, or sugars can be functionalized as ethers or esters. Moreover,the entire sugar moiety can be replaced with sterically andelectronically similar structures, such as aza-sugars and carbocyclicsugar analogs. Examples of modifications in a base moiety includealkylated purines and pyrimidines, acylated purines or pyrimidines, orother well-known heterocyclic substitutes. Nucleic acid monomers can belinked by phosphodiester bonds or analogs of such linkages. Nucleicacids can be either single stranded or double stranded.

By chimeric antigen receptor (CAR) is intended molecules that combine abinding domain against a component present on the target cell, forexample an antibody-based specificity for a desired antigen (e.g., tumorantigen) with a T cell receptor-activating intracellular domain togenerate a chimeric protein that exhibits a specific anti-targetcellular immune activity. Generally, CAR consists of an extracellularsingle chain antibody (scFv Fc) fused to the intracellular signalingdomain of the T cell antigen receptor complex zeta chain (scFv Fc:ζ) andhave the ability, when expressed in T cells, to redirect antigenrecognition based on the monoclonal antibody's specificity. One exampleof CAR used in the present invention is a CAR directing against CD123antigen and can comprise as non limiting example the amino acidsequences: SEQ ID NO: 23 to 48 and 160.

The term “endonuclease” refers to any wild-type or variant enzymecapable of catalyzing the hydrolysis (cleavage) of bonds between nucleicacids within a DNA or RNA molecule, preferably a DNA molecule.Endonucleases do not cleave the DNA or RNA molecule irrespective of itssequence, but recognize and cleave the DNA or RNA molecule at specificpolynucleotide sequences, further referred to as “target sequences” or“target sites”. Endonucleases can be classified as rare-cuttingendonucleases when having typically a polynucleotide recognition sitegreater than 12 base pairs (bp) in length, more preferably of 14-55 bp.Rare-cutting endonucleases significantly increase HR by inducing DNAdouble-strand breaks (DSBs) at a defined locus (Perrin, Buckle et al.1993; Rouet, Smih et al. 1994; Choulika, Perrin et al. 1995; Pingoud andSilva 2007). Rare-cutting endonucleases can for example be a homingendonuclease (Paques and Duchateau 2007), a chimeric Zinc-Fingernuclease (ZFN) resulting from the fusion of engineered zinc-fingerdomains with the catalytic domain of a restriction enzyme such as Fokl(Porteus and Carroll 2005), a Cas9 endonuclease from CRISPR system(Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong,Ran et al. 2013; Mali, Yang et al. 2013) or a chemical endonuclease(Eisenschmidt, Lanio et al. 2005; Arimondo, Thomas et al. 2006). Inchemical endonucleases, a chemical or peptidic cleaver is conjugatedeither to a polymer of nucleic acids or to another DNA recognizing aspecific target sequence, thereby targeting the cleavage activity to aspecific sequence. Chemical endonucleases also encompass syntheticnucleases like conjugates of orthophenanthroline, a DNA cleavingmolecule, and triplex-forming oligonucleotides (TFOs), known to bindspecific DNA sequences (Kalish and Glazer 2005). Such chemicalendonucleases are comprised in the term “endonuclease” according to thepresent invention.

By a “TALE-nuclease” (TALEN) is intended a fusion protein consisting ofa nucleic acid-binding domain typically derived from a TranscriptionActivator Like Effector (TALE) and one nuclease catalytic domain tocleave a nucleic acid target sequence. The catalytic domain ispreferably a nuclease domain and more preferably a domain havingendonuclease activity, like for instance I-Tevl, ColE7, NucA and Fok-I.In a particular embodiment, the TALE domain can be fused to ameganuclease like for instance I-Crel and 1-Onul or functional variantthereof. In a more preferred embodiment, said nuclease is a monomericTALE-Nuclease. A monomeric TALE-Nuclease is a TALE-Nuclease that doesnot require dimerization for specific recognition and cleavage, such asthe fusions of engineered TAL repeats with the catalytic domain ofI-Tevl described in WO2012138927. Transcription Activator like Effector(TALE) are proteins from the bacterial species Xanthomonas comprise aplurality of repeated sequences, each repeat comprising di-residues inposition 12 and 13 (RVD) that are specific to each nucleotide base ofthe nucleic acid targeted sequence. Binding domains with similar modularbase-per-base nucleic acid binding properties (MBBBD) can also bederived from new modular proteins recently discovered by the applicantin a different bacterial species. The new modular proteins have theadvantage of displaying more sequence variability than TAL repeats.Preferably, RVDs associated with recognition of the differentnucleotides are HD for recognizing C, NG for recognizing T, NI forrecognizing A, NN for recognizing G or A, NS for recognizing A, C, G orT, HG for recognizing T, IG for recognizing T, NK for recognizing G, HAfor recognizing C, ND for recognizing C, HI for recognizing C, HN forrecognizing G, NA for recognizing G, SN for recognizing G or A and YGfor recognizing T, TL for recognizing A, VT for recognizing A or G andSW for recognizing A. In another embodiment, critical amino acids 12 and13 can be mutated towards other amino acid residues in order to modulatetheir specificity towards nucleotides A, T, C and G and in particular toenhance this specificity. TALE-nuclease have been already described andused to stimulate gene targeting and gene modifications (Boch, Scholzeet al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et al. 2010;Li, Huang et al. 2011). Engineered TAL-nucleases are commerciallyavailable under the trade name TALEN™ (Cellectis, 8 rue de la CroixJarry, 75013 Paris, France).

The rare-cutting endonuclease according to the present invention canalso be a Cas9 endonuclease. Recently, a new genome engineering tool hasbeen developed based on the RNA-guided Cas9 nuclease (Gasiunas,Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran et al.2013; Mali, Yang et al. 2013) from the type II prokaryotic CRISPR(Clustered Regularly Interspaced Short palindromic Repeats) adaptiveimmune system (see for review (Sorek, Lawrence et al. 2013)). The CRISPRAssociated (Cas) system was first discovered in bacteria and functionsas a defense against foreign DNA, either viral or plasmid.CRISPR-mediated genome engineering first proceeds by the selection oftarget sequence often flanked by a short sequence motif, referred as theproto-spacer adjacent motif (PAM). Following target sequence selection,a specific crRNA, complementary to this target sequence is engineered.Trans-activating crRNA (tracrRNA) required in the CRISPR type II systemspaired to the crRNA and bound to the provided Cas9 protein. Cas9 acts asa molecular anchor facilitating the base pairing of tracRNA with cRNA(Deltcheva, Chylinski et al. 2011). In this ternary complex, the dualtracrRNA:crRNA structure acts as guide RNA that directs the endonucleaseCas9 to the cognate target sequence. Target recognition by theCas9-tracrRNA:crRNA complex is initiated by scanning the target sequencefor homology between the target sequence and the crRNA. In addition tothe target sequence-crRNA complementarity, DNA targeting requires thepresence of a short motif adjacent to the protospacer (protospaceradjacent motif—PAM). Following pairing between the dual-RNA and thetarget sequence, Cas9 subsequently introduces a blunt double strandbreak 3 bases upstream of the PAM motif (Garneau, Dupuis et al. 2010).

Rare-cutting endonuclease can be a homing endonuclease, also known underthe name of meganuclease. Such homing endonucleases are well-known tothe art (Stoddard 2005). Homing endonucleases recognize a DNA targetsequence and generate a single- or double-strand break. Homingendonucleases are highly specific, recognizing DNA target sites rangingfrom 12 to 45 base pairs (bp) in length, usually ranging from 14 to 40bp in length. The homing endonuclease according to the invention may forexample correspond to a LAGLIDADG endonuclease, to a HNH endonuclease,or to a GIY-YIG endonuclease. Preferred homing endonuclease according tothe present invention can be an I-Crel variant.

By “delivery vector” or “delivery vectors” is intended any deliveryvector which can be used in the present invention to put into cellcontact (i.e “contacting”) or deliver inside cells or subcellularcompartments (i.e “introducing”) agents/chemicals and molecules(proteins or nucleic acids) needed in the present invention. Itincludes, but is not limited to liposomal delivery vectors, viraldelivery vectors, drug delivery vectors, chemical carriers, polymericcarriers, lipoplexes, polyplexes, dendrimers, microbubbles (ultrasoundcontrast agents), nanoparticles, emulsions or other appropriate transfervectors. These delivery vectors allow delivery of molecules, chemicals,macromolecules (genes, proteins), or other vectors such as plasmids,peptides developed by Diatos. In these cases, delivery vectors aremolecule carriers. By “delivery vector” or “delivery vectors” is alsointended delivery methods to perform transfection.

The terms “vector” or “vectors” refer to a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked. A“vector” in the present invention includes, but is not limited to, aviral vector, a plasmid, a RNA vector or a linear or circular DNA or RNAmolecule which may consists of a chromosomal, non chromosomal,semi-synthetic or synthetic nucleic acids. Preferred vectors are thosecapable of autonomous replication (episomal vector) and/or expression ofnucleic acids to which they are linked (expression vectors). Largenumbers of suitable vectors are known to those of skill in the art andcommercially available.

Viral vectors include retrovirus, adenovirus, parvovirus (e. g.adenoassociated viruses), coronavirus, negative strand RNA viruses suchas orthomyxovirus (e. g., influenza virus), rhabdovirus (e. g., rabiesand vesicular stomatitis virus), paramyxovirus (e. g. measles andSendai), positive strand RNA viruses such as picornavirus andalphavirus, and double-stranded DNA viruses including adenovirus,herpesvirus (e. g., Herpes Simplex virus types 1 and 2, Epstein-Barrvirus, cytomegalovirus), and poxvirus (e. g., vaccinia, fowlpox andcanarypox). Other viruses include Norwalk virus, togavirus, flavivirus,reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.Examples of retroviruses include: avian leukosis-sarcoma, mammalianC-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus,spumavirus (Coffin, J. M., Retroviridae: The viruses and theirreplication, In Fundamental Virology, Third Edition, B. N. Fields, etal., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

By “lentiviral vector” is meant HIV-Based lentiviral vectors that arevery promising for gene delivery because of their relatively largepackaging capacity, reduced immunogenicity and their ability to stablytransduce with high efficiency a large range of different cell types.Lentiviral vectors are usually generated following transienttransfection of three (packaging, envelope and transfer) or moreplasmids into producer cells. Like HIV, lentiviral vectors enter thetarget cell through the interaction of viral surface glycoproteins withreceptors on the cell surface. On entry, the viral RNA undergoes reversetranscription, which is mediated by the viral reverse transcriptasecomplex. The product of reverse transcription is a double-strandedlinear viral DNA, which is the substrate for viral integration in theDNA of infected cells. By “integrative lentiviral vectors (or LV)”, ismeant such vectors as non limiting example, that are able to integratethe genome of a target cell. At the opposite by “non-integrativelentiviral vectors (or NILV)” is meant efficient gene delivery vectorsthat do not integrate the genome of a target cell through the action ofthe virus integrase.

Delivery vectors and vectors can be associated or combined with anycellular permeabilization techniques such as sonoporation orelectroporation or derivatives of these techniques.

By cell or cells is intended any eukaryotic living cells, primary cellsand cell lines derived from these organisms for in vitro cultures.

By “primary cell” or “primary cells” are intended cells taken directlyfrom living tissue (i.e. biopsy material) and established for growth invitro, that have undergone very few population doublings and aretherefore more representative of the main functional components andcharacteristics of tissues from which they are derived from, incomparison to continuous tumorigenic or artificially immortalized celllines.

As non-limiting examples cell lines can be selected from the groupconsisting of CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells,U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLacells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells; Molt 4cells.

All these cell lines can be modified by the method of the presentinvention to provide cell line models to produce, express, quantify,detect, study a gene or a protein of interest; these models can also beused to screen biologically active molecules of interest in research andproduction and various fields such as chemical, biofuels, therapeuticsand agronomy as non-limiting examples.

by “mutation” is intended the substitution, deletion, insertion of up toone, two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, fifteen, twenty, twenty five, thirty, forty,fifty, or more nucleotides/amino acids in a polynucleotide (cDNA, gene)or a polypeptide sequence. The mutation can affect the coding sequenceof a gene or its regulatory sequence. It may also affect the structureof the genomic sequence or the structure/stability of the encoded mRNA.

by “variant(s)”, it is intended a repeat variant, a variant, a DNAbinding variant, a TALE-nuclease variant, a polypeptide variant obtainedby mutation or replacement of at least one residue in the amino acidsequence of the parent molecule.

by “functional variant” is intended a catalytically active mutant of aprotein or a protein domain; such mutant may have the same activitycompared to its parent protein or protein domain or additionalproperties, or higher or lower activity.

“identity” refers to sequence identity between two nucleic acidmolecules or polypeptides. Identity can be determined by comparing aposition in each sequence which may be aligned for purposes ofcomparison. When a position in the compared sequence is occupied by thesame base, then the molecules are identical at that position. A degreeof similarity or identity between nucleic acid or amino acid sequencesis a function of the number of identical or matching nucleotides atpositions shared by the nucleic acid sequences. Various alignmentalgorithms and/or programs may be used to calculate the identity betweentwo sequences, including FASTA, or BLAST which are available as a partof the GCG sequence analysis package (University of Wisconsin, Madison,Wis.), and can be used with, e.g., default setting. For example,polypeptides having at least 70%, 85%, 90%, 95%, 98% or 99% identity tospecific polypeptides described herein and preferably exhibitingsubstantially the same functions, as well as polynucleotide encodingsuch polypeptides, are contemplated.

“similarity” describes the relationship between the amino acid sequencesof two or more polypeptides. BLASTP may also be used to identify anamino acid sequence having at least 70%, 75%, 80%, 85%, 87.5%, 90%,92.5%, 95%, 97.5%, 98%, 99% sequence similarity to a reference aminoacid sequence using a similarity matrix such as BLOSUM45, BLOSUM62 orBLOSUM80. Unless otherwise indicated a similarity score will be based onuse of BLOSUM62. When BLASTP is used, the percent similarity is based onthe BLASTP positives score and the percent sequence identity is based onthe BLASTP identities score. BLASTP “Identities” shows the number andfraction of total residues in the high scoring sequence pairs which areidentical; and BLASTP “Positives” shows the number and fraction ofresidues for which the alignment scores have positive values and whichare similar to each other. Amino acid sequences having these degrees ofidentity or similarity or any intermediate degree of identity ofsimilarity to the amino acid sequences disclosed herein are contemplatedand encompassed by this disclosure. The polynucleotide sequences ofsimilar polypeptides are deduced using the genetic code and may beobtained by conventional means. For example, a functional variant ofpTalpha can have 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%,99% sequence similarity to the amino acid sequence of SEQ ID NO: 107. Apolynucleotide encoding such a functional variant would be produced byreverse translating its amino acid sequence using the genetic code.

“signal-transducing domain” or “co-stimulatory ligand” refers to amolecule on an antigen presenting cell that specifically binds a cognateco-stimulatory molecule on a T-cell, thereby providing a signal which,in addition to the primary signal provided by, for instance, binding ofa TCR/CD3 complex with an MHC molecule loaded with peptide, mediates a Tcell response, including, but not limited to, proliferation activation,differentiation and the like. A co-stimulatory ligand can include but isnot limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL,OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, an agonist or antibodythat binds Toll ligand receptor and a ligand that specifically bindswith B7-H3. A co-stimulatory ligand also encompasses, inter alia, anantibody that specifically binds with a co-stimulatory molecule presenton a T cell, such as but not limited to, CD27, CD28, 4-IBB, OX40, CD30,CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2,CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the cell, such as, but notlimited to proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and Toll ligand receptor.

A “co-stimulatory signal” as used herein refers to a signal, which incombination with primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules.

The term “extracellular ligand-binding domain” as used herein is definedas an oligo- or polypeptide that is capable of binding a ligand.Preferably, the domain will be capable of interacting with a cellsurface molecule. For example, the extracellular ligand-binding domainmay be chosen to recognize a ligand that acts as a cell surface markeron target cells associated with a particular disease state. Thusexamples of cell surface markers that may act as ligands include thoseassociated with viral, bacterial and parasitic infections, autoimmunedisease and cancer cells.

The term “subject” or “patient” as used herein includes all members ofthe animal kingdom including non-human primates and humans.

The term “relapsed” refers to a situation where a subject or a mammal,who has had a remission of cancer after therapy has a return of cancercells.

The term “refractory or resistant” refers to a circumstance where asubject or a mammal, even after intensive treatment, has residual cancercells in his body.

The term “drug resistance” refers to the condition when a disease doesnot respond to the treatment of a drug or drugs. Drug resistance can beeither intrinsic (or primary resistance), which means the disease hasnever been responsive to the drug or drugs, or it can be acquired, whichmeans the disease ceases responding to a drug or drugs that the diseasehad previously responded to (secondary resistance). In certainembodiments, drug resistance is intrinsic. In certain embodiments, thedrug resistance is acquired.

The term “hematologic malignancy” or “hematologic cancer” refers to acancer of the body's blood-bone marrow and/or lymphatic tissue. Examplesof hematological malignancies include, for instance, myelodysplasia,leukemia, lymphomas, such as cutaneous Lymphomas, non-Hodgkin'slymphoma, Hodgkin's disease (also called Hodgkin's lymphoma), andmyeloma, such as acute lymphocytic leukemia (ALL), acute myeloidleukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocyticleukemia (CLL), chronic myeloid leukemia (CML), chronic neutrophilicleukemia (CNL), acute undifferentiated leukemia (AUL), anaplasticlarge-cell lymphoma (ALCL), prolymphocytic leukemia (PML), juvenilemyelomonocyctic leukemia (JMML), adult T-cell ALL, AML with trilineagemyelodysplasia (AML/TMDS), mixed lineage leukemia (MLL), myelodysplasticsyndromes (MDSs), myeloproliferative disorders (MPD), and multiplemyeloma (MM).

The term “leukemia” refers to malignant neoplasms of the blood-formingtissues, including, but not limited to, chronic lymphocytic leukemia orchronic lymphoid leukemia, chronic myelocytic leukemia, or chronicmyelogenous leukemia, acute lymphoblastic leukemia, acute myeloidleukemia or acute myelogenous leukemia (AML) and acute myeloblasticleukemia.

In general, a primary cell is a cell isolated from a blood sample or abiopsy and then optionally further cultured in vitro. A cell line is acellular culture of a transformed ie cancerous cell, preferably ahomogenous cellular culture of a transformed ie cancerous cell (whereina marker is represented by a Gaussian curve).

The above written description of the invention provides a manner andprocess of making and using it such that any person skilled in this artis enabled to make and use the same, this enablement being provided inparticular for the subject matter of the appended claims, which make upa part of the original description.

Where a numerical limit or range is stated herein, the endpoints areincluded. Also, all values and subranges within a numerical limit orrange are specifically included as if explicitly written out.

The above description is presented to enable a person skilled in the artto make and use the invention, and is provided in the context of aparticular application and its requirements. Various modifications tothe preferred embodiments will be readily apparent to those skilled inthe art, and the generic principles defined herein may be applied toother embodiments and applications without departing from the spirit andscope of the invention. Thus, this invention is not intended to belimited to the embodiments shown, but is to be accorded the widest scopeconsistent with the principles and features disclosed herein.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples, which areprovided herein for purposes of illustration only, and are not intendedto be limiting unless otherwise specified.

General Method

In general, the CD123 CAR T cells of the invention were prepared using Tcells purified from Buffy coat samples from different donors. Theprocess and products satisfies the requirement of the Good ManufacturingPractices (FDA 21 CFR and EU GMP).

The clinical essay was conducted under Good Clinical Practices (UK GCPor USA GCP)

-   Blood 2014; 124(21):4689

Preclinical Study

Primary T-Cell Cultures

T cells were purified from Buffy coat samples provided by EFS(Etablissement Français du Sang, Paris, France) using Ficoll gradientdensity medium. The PBMC layer was recovered and T cells were purifiedusing a commercially available T-cell enrichment kit. Purified T cellswere activated in X-Vivo™-15 medium (Lonza) supplemented with 20 ng/mLHuman IL-2, 5% Human, and Dynabeads Human T activator CD3/CD28 at abead:cell ratio 1:1 (Life Technologies).

CAR mRNA Transfection

Transfections were done at Day 4 or Day 11 after T-cell purification andactivation. 5 millions of cells were transfected with 15 μg of mRNAencoding the different CAR constructs. CAR mRNAs were produced using T7mRNA polymerase transfections done using Cytopulse technology, byapplying two 0.1 mS pulses at 3000V/cm followed by four 0.2 mS pulses at325V/cm in 0.4 cm gap cuvettes in a final volume of 200 μl of“Cytoporation buffer T” (BTX Harvard Apparatus). Cells were immediatelydiluted in X-Vivo™-15 media and incubated at 37° C. with 5% CO₂. IL-2was added 2 h after electroporation at 20 ng/mL.

T-Cell Transduction

Transduction of T-cells with recombinant lentiviral vectors expressionthe CAR was carried out three days after T-cell purification/activation.Lentiviral vectors were produced by Vectalys SA (Toulouse, France) bytransfection of genomic and helper plasmids in HEK-293 cells.Transductions were carried out at a multiplicity of infection of 5,using 10⁶ cells per transduction. CAR detection at the surface ofT-cells was done using a recombinant protein consisting on the fusion ofthe extracellular domain of the human CD123 protein together with amurine IgG1 Fc fragment (produced by LakePharma). Binding of thisprotein to the CAR molecule was detected with a PE-conjugated secondaryantibody (Jackson Immunoresearch) targeting the mouse Fc portion of theprotein, and analyzed by flow cytometry.

Degranulation Assay (CD107a Mobilization)

T-cells were incubated in 96-well plates (40,000 cells/well), togetherwith an equal amount of cells expressing various levels of the CD123protein. Co-cultures were maintained in a final volume of 100 μl ofX-Vivo™-15 medium (Lonza) for 6 hours at 37° C. with 5% CO₂. CD107astaining was done during cell stimulation, by the addition of afluorescent anti-CD107a antibody at the beginning of the co-culture,together with 1 μg/ml of anti-CD49d, 1 μg/ml of anti-CD28, and 1×Monensin solution. After the 6 h incubation period, cells were stainedwith a fixable viability dye and fluorochrome-conjugated anti-CD8 andanalyzed by flow cytometry. The degranulation activity was determined asthe % of CD8+/CD107a+ cells, and by determining the mean fluorescenceintensity signal (MFI) for CD107a staining among CD8+ cells.Degranulation assays were carried out 24 h after mRNA transfection.

IFN Gamma Release Assay

T-cells were incubated in 96-well plates (40,000 cells/well), togetherwith cell lines expressing various levels of the CD123 protein.Co-cultures were maintained in a final volume of 100 μl of X-Vivo™-15medium (Lonza) for 24 hours at 37° C. with 5% CO₂. After this incubationperiod the plates were centrifuged at 1500 rpm for 5 minutes and thesupernatants were recovered in a new plate. IFN gamma detection in thecell culture supernatants was done by ELISA assay. The IFN gamma releaseassays were carried by starting the cell co-cultures 24 h after mRNAtransfection.

Cytotoxicity Assay

T-cells were incubated in 96-well plates (100,000 cells/well), togetherwith 10,000 target cells (expressing CD123) and 10,000 control(CD123neg) cells in the same well. Target and control cells werelabelled with fluorescent intracellular dyes (CFSE or Cell Trace Violet)before co-culturing them with CAR+ T-cells. The co-cultures wereincubated for 4 hours at 37′C with 5% CO₂. After this incubation period,cells were labelled with a fixable viability dye and analyzed by flowcytometry. Viability of each cellular population (target cells orCD123neg control cells) was determined and the % of specific cell lysiswas calculated. Cytotoxicity assays were carried out 48 h after mRNAtransfection.

T-Cell Transduction

Transduction of T-cells with recombinant lentiviral vectors expressionthe CAR was carried out three days after T-cell purification/activation.CAR detection at the surface of T-cells was done using a recombinantprotein consisting on the fusion of the extracellular domain of thehuman CD123 protein, together with a murine IgG1 Fc fragment. Binding ofthis protein to the CAR molecule was detected with afluorochrome-conjugated secondary antibody targeting the mouse Fcportion of the protein, and analyzed by flow cytometry.

Anti-Tumor Mouse Model

Immuno-deficient NOG mice were intravenously (iv) injected with (CD123expressing_MOLM13-Luciferase cells as an AML xenograft mouse model.Optionally, mice received an anti-cancer treatment that is PNA or FLAG.Mice were then iv injected (either 2 or 7 days after injection of thetumor cell line) with different doses of CAR+ T-cells to be tested, orwith T-cells that were not transduced with the CAR lentiviral vector.Bioluminescent signals were determined at the day of T-cell injection(DO), at D7, 14, 21, 28 and 40 after T-cell injection in order to followtumoral progression on the different animals.

Clinical Study

A Phase I dose escalation trial is designed to evaluate the safety andthe biologic efficacy of allogeneic TCR KO specific cytotoxicT-lymphocytes (CTL) genetically modified to express artificial T-cellreceptors (CAR) targeting the CD123 molecule (CD123CAR) in patients whohave relapsed/refractory Acute myeloid Leukemia (AML), blasticplasmacytoid dendritic cell neoplasm, bridge to transplant.

Each patient will receive at least one dose of donor derived,genetically modified CTL and will be monitored for toxicity anddetection of transduced CTL as well as disease specific markers.

Any of the following may vary individually upon medical indication.

Condition Intervention Phase Acute myeloid Leukemia Biological: Phase 1Blastic plasmacytoid dendritic Biological/Genetically cell neoplasmModified T cells Bridge to transplant

-   Study Type: Interventional-   Study Design: Endpoint Classification: Safety/Efficacy Study    -   Intervention Model: Single Group Assignment    -   Masking: Open Label    -   Primary Purpose: Treatment-   Official Title: A Phase I Dose Escalation Trial Using In Vitro    Expanded Allogeneic Cytotoxic T-Lymphocytes (CTLs) Genetically    Targeted to the B-Cell Specific Antigen CD123 Positive Residual Or    Relapsed Acute myeloid Leukemia, Blastic plasmacytoid dendritic cell    neoplasm, during Bridge to transplant.    Key words Acute myeloid Leukemia, Blastic plasmacytoid dendritic    cell neoplasm, Bridge to transplant

Primary Outcome Measures:

Evaluate the safety/persistence of escalating doses of allogeneicspecific CTL modified to express artificial T cell receptors targetingCD123 molecule given for persistence or relapse of AML, for Blasticplasmacytoid dendritic cell neoplasm or in a Bridge to transplant.

Secondary Outcome Measures:

-   -   To assess the effects of the adoptively transferred CD123        specific T-cells on the progression of AML.    -   To quantitate the number of CD123 chimeric antigen receptor        (CD123 CAR) positive T-cells in the blood at defined intervals        post infusion in order to determine their survival and        proliferation in the host

To quantitate the number of CD123 chimeric antigen receptor (CD123 CAR)positive T-cells in the blood at defined intervals post infusion ofRituximab (375 mg/m2)

Arms Assigned interventions Experimental: Biological/ Biological:Biological/Genetically Genetically Modified T cells Modified T cellsPatients with persistent minimal Following completion of the residualdisease (+MRD) or chemotherapy, genetically relapsed AML will receive amodified T cells will be given conditioning chemotherapy intravenouslyat one of 3 dose regimen followed by intravenous levels (10⁵, 10⁶ and10⁷). After the infusion of allogeneic specific infusion patients willbe monitored cytotoxic T-cells (CTLs) clinically and with serial bloodand genetically modified ex vivo to marrow evaluations to assess expressthe CD123-specific toxicity, therapeutic effects, and chimericartificial T-cell receptor. the in-vivo survival of the geneticallymodified T-cells.

Eligibility Genders Eligible for Study: Both Accepts Healthy Volunteers:No Criteria Inclusion Criteria:

-   -   History of CD123+ leukemia with evidence of bone marrow relapse        or persistent.    -   Persistent minimal residual disease must be demonstrated by        morphology, FISH, flow cytometry or RT-PCR with at least 2        sequential testings separated by at least 1 week.    -   No age restriction for patients    -   KPS or Lansky score> or = to 40    -   Renal function (measured prior to conditioning chemotherapy)    -   Hepatic function (measured prior to conditioning chemotherapy):    -   AST≦5× the institutional ULN Elevation secondary to leukemic        involvement is not an exclusion criterion. Leukemic involvement        will be determined by the presence of progressive relapse        defined by escalating bone marrow or peripheral blood leukemia        blasts within the previous month and the absence of initiation        of know hepatotoxic medication (e.g. azoles).    -   Total bilirubin≦2.5× the institutional ULN    -   Adequate cardiac function (e.g. LVEF≧40%) as assessed by ECHO or        MUGA or other similar cardia imaging performed within 1 month of        enrollment.    -   Pulmonary function (measured prior to conditioning        chemotherapy):    -   Oxygen saturation≧90% on room air

Donor Eligibility:

-   -   The donor, including a third party donor, must consent to a        leukapheresis or whole blood donation(s) obtained at one or more        phlebotomies which, in aggregate, will total approximately 250        ml for adults and no more than 5 ml/kg per draw from pediatric        donors.    -   Related donors<18 years of age requiring placement of a        leukapheresis catheter will donate peripheral blood collected by        phlebotomy (including a unit of blood if weight permits) and        shall not undergo catheter placement for leukapheresis as this        is considered above minimal risk to the donor.    -   There is no upper age limit for a donor. However, the minimum        age for a related donor is 7 years as this is the youngest age a        person can be considered capable of giving assent to participate        in a research study.    -   Donor's high resolution HLA typing must be available for review    -   CBC within one week of donation. Results of tests must be within        a range that would not preclude donating blood or undergoing        leukapheresis.    -   Serologic testing for transmissible diseases will be performed        as per institutional guidelines adopted from extant NMDP and        FACT guidelines. Donors should be considered eligible to donate        leukapheresis or blood based on these guidelines (i.e. blood        donation guidelines)

Exclusion Criteria:

-   -   Patients with active HIV, hepatitis B or hepatitis C infection.    -   Patients with any concurrent active malignancies as defined by        malignancies requiring any therapy other than expectant        observation.    -   Females who are pregnant.    -   Patients will be excluded if they have isolated extra-medullary        relapse of ALL.    -   Patients with active (grade 2-4) acute graft versus host disease        (GVHD), chronic GVHD or an overt autoimmune disease (e.g.        hemolytic anemia) requiring glucocorticosteroid treatment (>0.5        mg/kg/day prednisone or its equivalent) as treatment    -   Active central nervous system (CNS) leukemia, as defined by        unequivocal morphologic evidence of lymphoblasts in the        cerebrospinal fluid (CSF) or symptomatic CNS leukemia (i.e.        cranial nerve palsies or other significant neurologic        dysfunction) within 28 days of treatment. Prophylactic        intrathecal medication is not a reason for exclusion.    -   Adult patients (≧18 years old) with the following cardiac        conditions will be excluded:    -   New York Heart Association (NYHA) stage III or IV congestive        heart failure    -   Myocardial infarction≦6 months prior to enrollment    -   History of clinically significant ventricular arrhythmia or        unexplained syncope, not believed to be vasovagal in nature or        due to dehydration.    -   History of severe non-ischemic cardiomyopathy with EF≦20%

The first data demonstrate that the engineered T cell of the inventioncan be infused iv at a dose of 10⁷ cells/kg to relapse refractory AMLpatients and selectively clear CD123-expressing cancerous cells for atleast 11 months.

EXAMPLES Example 1: Proliferation of TCRalpha Inactivated CellsExpressing a CD123-CAR

Heterodimeric TALE-nuclease targeting two 17-bp long sequences (calledhalf targets) separated by an 15-bp spacer within T-cell receptor alphaconstant chain region (TRAC) gene were designed and produced. Each halftarget is recognized by repeats of the half TALE-nucleases listed inTable 10.

TABLE 10 TAL-nucleases targeting TCRalpha gene Target Target sequenceRepeat sequence Half TALE-nuclease TRAC_T01 TTGTCCCACAGATATCC RepeatTRAC_T01-L TRAC_T01-L TALEN Agaaccctgaccctg (SEQ ID NO: 14) (SEQ ID NO:16) CCGTGTACCAGCTGAGA Repeat TRAC_T01-R TRAC_T01-R TALEN (SEQ ID NO: 13)(SEQ ID NO: 15) (SEQ ID NO: 17)

Each TALE-nuclease construct was subcloned using restriction enzymedigestion in a mammalian expression vector under the control of the T7promoter. mRNA encoding TALE-nuclease cleaving TRAC genomic sequencewere synthesized from plasmid carrying the coding sequence downstreamfrom the T7 promoter.

Purified T cells preactivated during 72 hours with antiCD3/CD28 coatedbeads were transfected with each of the 2 mRNAs encoding both halfTRAC_T01 TALE-nucleases. 48 hours post-transfection, different groups ofT cells from the same donor were respectively transduced with alentiviral vector encoding one of the CD-123 CAR previously described(SEQ ID NO: 23 to 48). 2 days post-transduction, CD3_(NEG) cells werepurified using anti-CD3 magnetic beads and 5 days post-transductioncells were reactivated with soluble anti-CD28 (5 μg/ml).

Cell proliferation was followed for up to 30 days after reactivation bycounting cell 2 times per week. Increased proliferation in TCR alphainactivated cells expressing the CD-123 CARs, especially whenreactivated with anti-CD28, was observed compared to non-transducedcells.

To investigate whether the human T cells expressing the CD123-CARdisplay activated state, the expression of the activation marker CD25are analyzed by FACS 7 days post transduction. The purified cellstransduced with the lentiviral vector encoding CD-123 CAR assayed forCD25 expression at their surface in order to assess their activation incomparison with the non-transduced cells. Increased CD25 expression isexpected both in CD28 reactivation or no reactivation conditions.

Example 2

Construction of CD123 CAR Using Anti-CD123 scFv Antibody FragmentsDerived from Klon43, Functional Analysis in TCR KO and dCK KO TCR CD123Expressing Cells

An scFv from Klon 43 or humanized scFv was prepared using a combinationof a VH (SEQ ID NO 12, a L (SEQ ID NO 10), a VL SEQ ID NO 11, or a VHselected from SEQ ID NO 24 to SEQ ID NO. 30 a linker L and a VL selectedfrom SEQ ID NO. 18 to SEQ ID NO. 23, respectively was used to generateCD123 Chimeric Antigen Receptors (CD123 CARs of the invention) and toscreen them to select performant (with anti-tumoral activity with theless side effects).

Architectures V1 or V3, preferably V3 were used (FIG. 2 and FIG. 3) andactivity was determined upon expression in human T-cells (FIG. 4, FIG.5, FIG. 6 and FIG. 7).

The results illustrated in FIG. 4 shows degranulation activity ofdifferent a scFv according to the invention for one architecture (v3:CD8-hinge/CD8-transmembrane), when CAR+ T-cells were co-cultured for 6hours with CD123 expressing cells (RPM18226), or with cells that do notexpress CD123 (K562). White bars correspond to degranulation signalsobserved in T-cells that were cultured alone, black bars represent thesignals observed when T-cells were co-cultured with RPM18226 cells, andgray bars show degranulation signals of T-cells co-cultured with K562cells.

FIG. 5 shows the amount of IFN gamma released by T-cells whenco-cultured for 24 h with cells expressing different levels of CD123(KG1a or RPM18226), or with cells that do not express CD123 (K562). IFNgamma release from T-cells cultured alone, in the same conditions thatthe co-cultures, is also shown. The experiments were done for threeindependent donors, and results from a representative donor are shownhere.

FIGS. 6 and 7 show a dose-response of the specific cytolytic activity ofCAR-T cells in vivo in mice treated with PNA (20 mg/kg) ip.

Immunodefficient mice were injected with MOLM13-Luciferase cells 2 daysbefore injection of non-transduced human T-cells, or with differentdoses of anti-CD123 CAR+ T-cells. The results represent thebioluminescent signal observed at different time points after T-cellinjection.

Example 3: Clinical Study

Clinical study provided data demonstrating the feasibility of theclinical study described herein.

At least one patient suffering refractory/relapsed AML received atreatment according to the invention that significantly increased herlife expectancy by more than 16 months and reduced the cancerous cellsbelow detection level for at least 6 months.

Engineered immune cells expressing the CD123 were detected at least 3months after infusion.

Administration of Rituximab (Rituxan®).

The total dose of Rituximab administered by intravenous route during 4day after an initial rituximab dose of 375 mg/m2 was 2,250 mg/m2.

Premedication was performed before each infusion with acetaminophen andan antihistamine.

In another embodiment, the first Infusion was initiated at a rate of 50mg/hr. In the absence of infusion toxicity, infusion rate was 50 mg/hrincremented every 30 minutes, to a maximum of 400 mg/hr.

For the subsequent Infusions, it was performed at a rate of 100 mg/hr.In the absence of infusion toxicity, increased to a maximum of 400 mg/hrto reach 2,250 mg/m2.

The results show that the level of CD123 CAR cells in the blood ofpatients was below detection at 24 h after the last infusion.

Klo43-1 (SEQ ID NO. 31)

ISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

In one embodiment Klon43-1 is

PLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Klo43-3 (SEQ ID NO. 32)

QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR

In one embodiment Klon43-3 is

LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR Klo43-5(SEQ ID NO. 33)

VAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR

In one embodiment Klon43-5 is

SVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STATKDTYDALHMQALPPR

The Humanized CD123 CAR of the invention comprise one of the followingsequences:

Version 1 VH1 VL1MALPVTALLLPLALLLHAARPEVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL2MALPVTALLLPLALLLHAARPEVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL3MALPVTALLLPLALLLHAARPEVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL4MALPVTALLLPLALLLHAARPEVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL5MALPVTALLLPLALLLHAARPEVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL6MALPVTALLLPLALLLHAARPEVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Version1 VH2 VL1MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL2MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL3MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL4MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL5MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL6MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Version1 VH3 VL1MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL2MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL3MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL4MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL5MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL6MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Version1 VH4 VL1MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL2MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL3MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL4MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL5MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL6MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Version1 VH5 VL1MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL2MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL3MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL4MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL5MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL6MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Version1 VH6 VL1MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL2MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL3MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL4MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL5MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL6MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Version1 VH7 VL1MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL2MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL3MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL4MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL5MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL6MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Version3 VH1 VL1MALPVTALLLPLALLLHAARPEVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL2MALPVTALLLPLALLLHAARPEVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL3MALPVTALLLPLALLLHAARPEVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL4MALPVTALLLPLALLLHAARPEVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL5MALPVTALLLPLALLLHAARPEVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL6MALPVTALLLPLALLLHAARPEVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Version 3 VH2 VL1MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL2MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL3MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL4MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL5MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL6MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Version 3 VH3 VL1MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL2MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL3MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL4MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL5MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL6MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Version 3 VH4 VL1MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL2MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL3MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL4MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL5MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL6MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VH5 Version 3 VH5 VL1MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL2MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL3MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL4MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL5MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL6MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Version 3 VH6 VL1MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL2MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL3MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL4MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL5MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL6MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VH7 Version 3 VH7 VL1MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL2MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL3MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL4MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL5MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR VL6MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Version 5 VH1 VL1MALPVTALLLPLALLLHAARPEVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL2MALPVTALLLPLALLLHAARPEVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL3MALPVTALLLPLALLLHAARPEVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL4MALPVTALLLPLALLLHAARPEVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL5MALPVTALLLPLALLLHAARPEVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL6MALPVTALLLPLALLLHAARPEVKLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC Version 5 VH2VL1 MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL2MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL3MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL4MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL5MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL6MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC Version 5 VH3VL1 MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL2MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL3MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL4MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL5MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL6MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC Version 5 VH4VL1 MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL2MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL3MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL4MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL5MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL6MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYSASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VH5 Version 5VH5 VL1MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL2MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL3MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL4MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL5MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL6MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC Version 5 VH6VL1 MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL2MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL3MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL4MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL5MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL6MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VH7 Version 5VH7 VL1MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL2MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL3MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL4MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL5MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC VL6MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGFIRSKADGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSMADYKDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKLLIYSASYGQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYC

The following sequences are contemplated as preferred CAR of theinvention

(SEQ ID NO 172)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. (SEQ ID NO 173)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCKASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. (SEQ ID NO 174)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. (SEQ ID NO 175)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. (SEQ ID NO 176)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. (SEQ ID NO 177)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCKASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPDRFSGSGSGTDFTLTISSLQPEDLATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. (SEQ ID NO 178)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. (SEQ ID NO 179)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. (SEQ ID NO 180)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. (SEQ ID NO181) EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCKASQNVDSAVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. (SEQ ID NO182) EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. (SEQ ID NO183) EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. (SEQID NO 184)EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. (SEQ ID NO185) EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYAASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCKASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPDRFSGSGSGTDFTLTISSLQPEDLATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. (SEQ ID NO186) EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSQSIAYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. (SEQ ID NO187) EVQLVESGGGLVQPGRSLRLSCTASGFTFTDYYMSWVRQAPGKGLEWVGLIRSKADGYTTEYSASVKGRFTISRDDSKSILYLQMNSLKTEDTAVYYCARDAAYYSYYSPEGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPSSVSASVGDRVTITCRASQNVDSAVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGRGSGTDFTLTISSLQPEDFATYYCQQYYSTPWTFGQGTKVEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

According to the present invention these anti-CD123 CAR can include atleast one mimotope.

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1. The CD123 specific chimeric antigen receptor (CAR) comprising anextracellular domain comprising an extra cellular ligand binding-domaincomprising successively, a VH optionally humanized, a linker, a VLoptionally humanized, and a hinge, a transmembrane domain, a cytoplasmicdomain, and at least one epitope specific for a monoclonal antibody(mimotope).
 2. The CD123 CAR according to claim 1 comprising anextracellular domain comprising an extra cellular ligand binding-domaincomprising successively, a VH of SEQ ID NO 12, SEQ ID NO 24, SEQ ID NO25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29 or SEQ ID NO30, optionally humanized, a linker, a VL of SEQ ID NO 11, SEQ ID NO 18,SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22 or SEQ ID NO 23,optionally humanized, and a hinge, a transmembrane domain from CD8alpha, and a cytoplasmic domain including a CD3 zeta signaling domainand a co-stimulatory domain from 4-1BB.
 3. The CD123 CAR according toclaim 1, wherein the CAR lacks a sequence having identity to human CD28NP_006130.1.
 4. The CD123 CAR according to claim 1 comprising a sequenceof SEQ ID NO 172, SEQ ID NO 173, SEQ ID NO 174, SEQ ID NO 175, SEQ ID NO176, SEQ ID NO 177, SEQ ID NO 178, SEQ ID NO 179, SEQ ID NO 180, SEQ IDNO 181, SEQ ID NO 182, SEQ ID NO 183, SEQ ID NO 184, SEQ ID NO 185, SEQID NO 186 and SEQ ID NO 187, optionally further comprising at least oneSEQ ID NO
 161. 5. The CD123 CAR according to claim 1, wherein saidextracellular ligand binding-domain comprises at least one epitopespecific for a monoclonal antibody (mimotope) of SEQ ID NO 161, SEQ IDNO 162, SEQ ID NO 163, SEQ ID NO 164, SEQ ID NO 165, SEQ ID NO 166, SEQID NO 167, SEQ ID NO 168, SEQ ID NO 169 or SEQ ID NO 170, preferably ofSEQ ID NO 161 and of SEQ ID NO
 169. 6. The CD123 CAR according to claim1 comprising a sequence of SEQ ID NO 160, SEQ ID NO 171, SEQ ID NO 188,SEQ ID NO 189, SEQ ID NO 190, SEQ ID NO 191, SEQ ID NO 192, SEQ ID NO193, SEQ ID NO 194, SEQ ID NO 195, SEQ ID NO 196, or SEQ ID NO
 197. 7. Apolynucleotide encoding the CD123 specific CAR according to claim
 1. 8.An expression vector comprising the polynucleotide according to claim 7.9. An expression vector comprising a backbone and at least one sequenceencoding the CAR according to claim
 6. 10. An expression vectorcomprising a backbone, an EF1 promotor, an RQR8 open reading frame (RQR8ORF), a sequence coding any one of the CD123 CAR of claim
 6. 11. Anengineered immune cell comprising a T Cell Receptor (TCR) knock-out (KO)or TCR and human deoxycytidine kinase (dCK) KO and a CD123 specific CARaccording to claim 1, wherein the CAR is expressed on the cell surfaceof the immune cell.
 12. An engineered immune cell comprising apolynucleotide coding a CD123 specific CAR according to claim
 7. 13. Theengineered immune cell according to claim 11 further expressing asuicide domain at the cell surface.
 14. The engineered immune cellaccording to claim 11, wherein the cell suppresses expression of atleast one MHC protein.
 15. A method of treating a disease or conditionassociated with CD123 comprising administering to a subject in needthereof the engineered immune cell according to claim
 11. 16. The methodof treating according to claim 15, wherein the disease or condition isacute myelogenous leukemia (AML), BPDNL, or during or before bone marrowtransplantation.
 17. The method according to claim 15, wherein thedisease or condition is a lymphoproliferative disorder, acutemyelogenous leukemia, chronic myelogenous leukemia, myelodysplasticsyndrome, acute lymphoid leukemia, chronic lymphoid leukemia,myelodysplastic syndrome, or BPDNL.
 18. The CD123 specific CAR accordingto claim 1, wherein the linker is a linker of sequence (GGGGS)n, wherein“n” is 1-4.
 19. The CD123 specific CAR according to claim 18, wherein“n” is
 3. 20. The CD123 specific CAR according to claim 5, wherein theepitope specific for a monoclonal antibody is SEQ ID NO 161 or
 169. 21.The method of treating according to claim 16, wherein the AML isrefractory AML or relapsed AML.
 22. The method of treating according toclaim 17, wherein the lymphoproliferative disorder is leukemia orlymphoma.